Methods and compositions for modulating cancer stem cells

ABSTRACT

Disclosed are compositions and methods that use lysine demethylase inhibitors for inhibiting the growth of cancer stem cells or tumor initiating cells, for enhancing the biological effects of chemotherapeutic drugs or irradiation on cancer cells and/or for preventing cancer recurrence.

RELATED APPLICATIONS

This application claims priority to Australian Provisional ApplicationNo. 2013902309 entitled “Stem cell modulation”, filed on 25 Jun. 2013,and to Australian Provisional Application No. 2014900953 entitled “Stemcell modulation I”, filed on 19 Mar. 2014, the subject matter of each ofwhich is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to lysine demethylase inhibitors inmethods and composition for inhibiting the growth of cancer stem cellsor tumor initiating cells, for enhancing the biological effects ofchemotherapeutic drugs or irradiation on cancer cells and/or forpreventing cancer recurrence.

BACKGROUND OF THE INVENTION

Cancer stem cells (CSCs), or ‘precursor’ metastatic cells, initiatetumors and drive malignant progression by generating and supportingreplication of more differentiated non-stem cell progeny (see, forexample, Kleffel et al., 2013. Adv Exp Med Biol. 734:145-79; Chen etal., 2013. Acta Pharmacologica Sinica 34:732-740; Páez et al., 2012,Clin Cancer Res. 18(3):645-53). CSCs have been demonstrated to befundamentally responsible for tumorigenesis, cancer metastasis, tumorrelapse, drug resistance, and chemo- and radio-therapy failure.Unfortunately, the mechanisms by which CSCs cause tumor formation andgrowth and the potential role of CSC-specific differentiation plasticityin tumorigenicity are currently unknown.

Of interest, CSCs share many similar traits with normal stem cells. Forexample, CSCs have self-renewal capacity, namely, the ability to giverise to additional tumorigenic cancer stem cells, typically at a slowerrate than other dividing tumor cells, as opposed to a limited number ofdivisions. CSCs also have the ability to differentiate into multiplecell types (i.e., they are multipotent), which would explainhistological evidence that not only many tumors contain multiple celltypes native to the host organ, but also that heterogeneity is commonlyretained in tumor metastases.

CSCs express certain cell surface markers as listed for example in Table1

TABLE 1 CSC markers for distinct solid tumor types Breast Colon GliomaLiver Lung Melanoma Ovarian Pancreatic Prostate ABCB5 ALDH1 ALDH1 ABCG2CD24 β-catenin CD15 CD13 ALDH1 ABCB5 ALDH1 ALDH1 activity CD44 CD24 CD90CD24 ABCG2 ALDH1 CD24 CD24 CD44 CD90 CD26 CD133 CD44 CD90 CD20 CD44 CD44CD133 CD133 CD29 α₆ CD90 CD117 CD133 CD117 CD133 α₂ β₁ integrin integrinHedgehog- CD44 Nestin CD133 CD133 CD271 CD133 c-Met α₆ integrin Gliactivity α₆ integrin CD133 CXCR4 Trop2 CD166 Nestin LGR5 Nodal-Activin

Normal somatic stem cells are naturally resistant to chemotherapeuticagents—they have various pumps (such as multi-drug resistance (MDR)proteins) that pump out drugs, and efficient DNA repair mechanisms.Further, they also have a slow rate of cell turnover whilechemotherapeutic agents target rapidly replicating cells. CSCs, beingthe mutated counterparts of normal stem cells, may also have similarmechanisms that allow them to survive drug therapies and radiationtreatment. In other words, conventional chemotherapies andradiotherapies kill differentiated or differentiating cells, which formthe bulk of the tumor that are unable to regenerate tumors. Thepopulation of CSCs that gave rise to the differentiated anddifferentiating cells, on the other hand, could remain untouched andcause a relapse of the disease. A further danger for the conventionalanti-cancer therapy is the possibility that the treatment of, forinstance, chemotherapy, leaves only chemotherapy-resistant CSCs, and theensuing recurrent tumor will likely also be resistant to chemotherapy.

Consequently, there is a pressing need for the identification of novelapproaches that target cytotoxic drug-resistant, tumor-initiating CSCsfor preventing and/or treating disease recurrence and distant metastaticspread.

SUMMARY OF THE INVENTION

The present invention is based in part on the determination that histonedemethylases, including lysine specific demethylases (LSDs) (e.g., LSD1and LSD2), are overexpressed in breast CSCs and that inhibition of theseenzymes results in specific cell death of the breast CSCs. Based on thedetermination that LSDs are overexpressed in prostate, lung and bladderCSCs, the present inventors propose that other CSCs can also be treatedwith LSD inhibitors to reduce or inhibit their proliferation and/or tostimulate their death.

Accordingly, in one aspect, the present invention provides methods forinhibiting the proliferation, survival or viability of a CSC. Thesemethods generally comprise, consist or consist essentially of contactingthe CSC with a proliferation-, survival- or viability-inhibiting amountof a LSD inhibitor. In some embodiments, the CSC is selected frombreast, prostate, lung, bladder, pancreatic, colon, melanoma, liver orglioma CSCs.

In specific embodiments, the CSC is a breast cancer CSC. Suitably, theCSC has impaired or abrogated expression of the pluripotent stem cellmarkers Oct4 or Sox2 or expresses one or both of those markers at alevel or functional activity that is less than about ⅕, 1/10, 1/20,1/50, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰,10⁻¹¹, 10⁻¹², 10⁻¹³, 10⁻¹⁴ or about 10⁻¹⁵ of the level or functionalactivity of those markers on a pluripotent stem cell.

Suitably, the CSC expresses one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 ormore) CSC markers selected from ABCB5, ALDH1, ABCG2, α₆ integrin, σ₂ β₁integrin, β-catenin activity, CD15, CD13, CD20, CD24, CD26, CD29, CD44,CD90, CD133, CD166, CD271, c-Met, Hedgehog-Gli, Nestin, CXCR4, LGR5,Trop2 and Nodal-Activin. In some embodiments, the CSC expresses one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) CSC markers selected fromALDH1, CD24, CD44, CD90, CD133, Hedgehog-Gli, α₆ integrin. Inillustrative examples of this type, the CSC expresses CD24 and CD44(e.g., CD44^(high), CD24^(low).

Non-limiting examples of suitable LSD inhibitors include nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic (carbon containing) or inorganic molecules. In specificembodiments, the LSD inhibitor is selected from small moleculeinhibitors and nucleic acid molecules (e.g., ones that inhibit thetranscription or translation of a LSD gene (e.g., LSD1 or LSD2) or thatmediate RNA interference). In some embodiments, the LSD inhibitorreduces the expression of the LSD gene or the level or functionalactivity (e.g., reduces the level of a LSD polypeptide or reducesLSD-mediated demethylation) of a LSD inhibitor expression product toless than about 9/10, ⅘, 7/10, ⅗, ½, ⅖, 3/10, ⅕, 1/10, 1/20, 1/50, 10⁻¹,10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹²,10⁻¹³, 10⁻¹⁴ or about 10⁻¹⁵ of the expression of the LSD gene, or thelevel or functional activity of a corresponding LSD expression productin the absence of the inhibitor. In some embodiments, the LSD inhibitoris a selective LSD inhibitor (e.g., a selective LSD1 inhibitor or aselective LSD2 inhibitor). In other embodiments, the LSD inhibitor is aPan-LSD inhibitor. In still other embodiments, the LSD inhibitor is anon-selective LSD inhibitor.

In some embodiments, the methods for inhibiting the proliferation,survival or viability of the CSC further comprise detectingoverexpression of a LSD gene (e.g., LSD1 or LSD2) in the CSC prior tocontacting the CSC with the LSD inhibitor.

Suitably, the methods for inhibiting the proliferation, survival orviability of the CSC further comprise detecting that the CSC expressesone or more CSC markers as broadly described above.

In another aspect, the present invention provides methods for treatingor preventing a cancer (e.g., a metastatic cancer) in a subject, whereinthe cancer comprises CSCs and non-CSC tumor cells. These methodsgenerally comprise, consist or consist essentially of administering tothe subject a LSD inhibitor in an effective amount to inhibit theproliferation, survival or viability of the CSCs. In advantageousembodiments, the methods further comprise identifying that the subjecthas or is at risk of developing a cancer comprising CSCs and non-CSCtumor cells prior to the administration of the LSD inhibitor. In someembodiments, the cancer is selected from breast, prostate, lung,bladder, pancreatic, colon, melanoma, liver or glioma cancer. Suitably,the CSCs give rise to non-CSC tumor cells that are hormone-resistant. Inillustrative examples of this type, the non-CSC tumor cells have reducedor abrogated expression of one or more (e.g., 1 or 2) hormone receptorsselected from an estrogen receptor (ER) and a progesterone receptor(PR). Suitably, the cancer is selected from breast, prostate, lung,bladder, pancreatic, colon, melanoma, liver or brain cancer. In specificembodiments, the cancer is breast cancer.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting overexpression of a LSD gene (e.g., LSD1 orLSD2) in a tumor sample obtained from the subject, wherein the tumorsample comprises the CSCs, prior to administering the LSD inhibitor tothe subject.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting expression of one or more CSC markers asbroadly described above in a tumor sample obtained from the subject,wherein the tumor sample comprises the CSCs, prior to administering theLSD inhibitor to the subject.

Yet another aspect of the present invention provides methods fortreating or preventing a cancer (e.g., a metastatic cancer) in asubject, wherein the cancer comprises CSCs and non-CSC tumor cells.These methods generally comprise, consist or consist essentially ofconcurrently administering to the subject a LSD inhibitor in aneffective amount to inhibit the proliferation, survival or viability ofthe CSCs and a cancer therapy or agent that inhibits the proliferation,survival or viability of the non-CSC tumor cells, to thereby treat orprevent the cancer. In some embodiments, the cancer therapy or agent isselected from radiotherapy, surgery, chemotherapy, hormone ablationtherapy, pro-apoptosis therapy and immunotherapy. In illustrativeexamples of this type, the cancer therapy or agent targets rapidlydividing cells or disrupts the cell cycle or cell division. Suitably,the methods further comprise identifying that the subject has or is atrisk of developing a cancer comprising CSCs and non-CSC tumor cellsprior to the co-administration. In some embodiments, the cancer isselected from breast, prostate, lung, bladder, pancreatic, colon,melanoma, liver or glioma cancer. Suitably, the non-CSC tumor cells arehormone-resistant. In illustrative examples of this type, the non-CSCtumor cells have reduced or abrogated expression of one or more (e.g., 1or 2) hormone receptors selected from an estrogen receptor (ER) and aprogesterone receptor (PR). Suitably, the cancer is selected frombreast, prostate, lung, bladder, pancreatic, colon, melanoma, liver orbrain cancer. In specific embodiments, the cancer is breast cancer.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting overexpression of a LSD gene (e.g., LSD1 orLSD2) in a tumor sample obtained from the subject, wherein the tumorsample comprises the CSCs and optionally the non-CSC tumor cells, priorto administering the LSD inhibitor to the subject.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting that the CSCs express one or more CSC markersas broadly described above prior to administering the LSD inhibitor tothe subject.

Suitably, the LSD inhibitor and the cancer therapy agent areadministered in synergistically effective amounts.

Typically, one or both of the LSD inhibitor and the cancer therapy oragent are administered on a routine schedule, for example, every day, atleast twice a week, at least three times a week, at least four times aweek, at least five times a week, at least six times a week, every week,every other week, every third week, every fourth week, every month,every two months, every three months, every four months, and every sixmonths.

In some embodiments, the cancer therapy is likely to expose the subjectto a higher risk of infection. Accordingly, in these embodiments, themethods may further comprise administering simultaneously, sequentiallyor separately with the LSD inhibitor and/or the cancer therapy/agent atleast one anti-infective agent that is effective against an infectionthat develops or that has an increased risk of developing byadministration of the cancer therapy, wherein the anti-infective agentis selected from antimicrobials, antibiotics, antivirals, antifungals,anthelmintics, antiprotozoals and nematocides.

In yet another aspect, the invention provides methods for identifyingagents that are useful for inhibiting proliferation, survival orviability of a CSC or for treating or preventing a cancer in a subject,wherein the cancer comprises CSCs. These methods generally comprisecontacting a preparation with a test agent, wherein the preparationcomprises (i) a polypeptide comprising an amino acid sequencecorresponding to at least a biologically active fragment of a LSD (e.g.,LSD1 or LSD2), or to a variant or derivative thereof; or (ii) apolynucleotide comprising a nucleotide sequence from which a transcriptof a LSD gene (e.g., LSD1 or LSD2) or portion thereof is producible, or(iii) a polynucleotide comprising at least a portion of a geneticsequence (e.g., a transcriptional element) that regulates the expressionof a LSD gene (e.g., LSD1 or LSD2), which is operably linked to areporter gene. A detected reduction in the level and/or functionalactivity of the polypeptide, transcript or transcript portion or anexpression product of the reporter gene, relative to a normal orreference level and/or functional activity in the absence of the testagent, indicates that the agent is useful for inhibiting proliferation,survival or viability of a CSC or for treating or preventing the cancer.

Still another aspect of the present invention provides methods ofproducing an agent for inhibiting proliferation, survival or viabilityof a CSC or for treating or preventing a cancer that comprises CSCs, asbroadly described above. These methods generally comprise: testing anagent suspected of inhibiting a LSD (e.g., LSD1 or LSD2) as broadlydescribed above; and synthesizing the agent on the basis that it testspositive for the inhibition. Suitably, the method further comprisesderivatizing the agent, and optionally formulating the derivatized agentwith a pharmaceutically acceptable carrier and/or diluent, to improvethe efficacy of the agent for inhibiting proliferation, survival orviability of a CSC or for treating or preventing a cancer that comprisesCSCs.

Another aspect of the present invention provides pharmaceuticalcompositions for inhibiting proliferation, survival or viability of aCSC or for treating or preventing a cancer that comprises CSCs andnon-CSC tumor cells, as broadly described above. These compositionsgenerally comprise, consist or consist essentially of a LSD inhibitor(e.g., LSD1 or LSD2 inhibitor) and an agent that inhibits theproliferation, survival or viability of the non-CSC tumor cells.

In a further aspect, the present invention provides the use of a LSDinhibitor (e.g., LSD1 or LSD2 inhibitor) for inhibiting proliferation,survival or viability of a CSC or for treating or preventing a cancercomprising CSCs, as broadly described above.

Still another aspect of the present invention provides the use of a LSDinhibitor (e.g., LSD1 or LSD2 inhibitor) for enhancing the efficacy of acancer therapy or agent that inhibits the proliferation, survival orviability of the non-CSC tumor cells.

In yet another aspect, the present invention provides the use of LSDinhibitor (e.g., LSD1 or LSD2 inhibitor) and a cancer therapy or agentthat inhibits the proliferation, survival or viability of the non-CSCtumor cells for treating or preventing a cancer that comprises CSCs andnon-CSC tumor cells, as broadly described above. In some embodiments,the LSD inhibitor and optionally the cancer therapy or agent areprepared or manufactured as medicaments for this purpose.

The present inventors have also found that it is possible to inhibit EMTof LSD-overexpressing non-CSC breast tumor cells and to inducemesenchymal-to-epithelial cell transition (MET) of breast CSCs byinhibiting the activity of LSDs (e.g., LSD1 and LSD2). Accordingly, itis proposed that LSD inhibitors are broadly useful for reducing orinhibiting the proliferation and EMT of LSD-overexpressing non-CSC tumorcells, and for inhibiting the proliferation, stimulating the death,and/or inducing MET of CSCs.

Thus, in another aspect, the present invention provides methods foraltering at least one of: (i) formation; (ii) proliferation; (iii)survival; (iv) viability; (v) maintenance; (vi) EMT; or (vii) MET of aLSD (e.g., LSD1 or LSD2)-overexpressing cell. These methods generallycomprise, consist or consist essentially of contacting theLSD-overexpressing cell with a formation-, proliferation-, survival-,viability-, maintenance-; EMT- or MET-modulating amount of a LSD (e.g.,LSD1 or LSD2) inhibitor. Suitably, the LSD-overexpressing cell isselected from a CSC and a non-CSC tumor cell, illustrative examples ofwhich include breast, prostate, lung, bladder, pancreatic, colon,melanoma, liver or glioma CSC and non-CSC tumor cells. In someembodiments, the CSC is a breast CSC (e.g., a breast epithelial CSC,including a breast ductal epithelial CSC). In some embodiments, thenon-CSC tumor cell is a breast non-CSC tumor cell (e.g., a breastepithelial non-CSC tumor cell, including a breast ductal epithelialnon-CSC tumor cell). Suitably, the LSD-overexpressing cell is contactedwith one or more of a LSD-overexpressing cell formation-inhibiting,proliferation-inhibiting, survival-inhibiting, viability-inhibiting,maintenance-inhibiting, EMT-inhibiting amount orMET-stimulating/inducing amount of the LSD inhibitor.

In some embodiments in which the LSD-overexpressing cell is a CSC, theCSC expresses one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) CSCmarkers selected from ABCB5, ALDH1, ABCG2, α₆ integrin, α₂ β₁ integrin,β-catenin activity, CD15, CD13, CD20, CD24, CD26, CD29, CD44, CD90,CD133, CD166, CD271, c-Met, Hedgehog-Gli, Nestin, CXCR4, LGR5, Trop2 andNodal-Activin. In some embodiments, the CSC expresses one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8 or more) CSC markers selected from ALDH1, CD24,CD44, CD90, CD133, Hedgehog-Gli, α₆ integrin. In illustrative examplesof this type, the CSC expresses CD24 and CD44 (e.g., CD44^(high),CD24^(low).

In some embodiments, the methods for altering at least one of: (i)formation; (ii) proliferation; (iii) survival; (iv) viability; (v)maintenance; (vi) EMT; or (vii) MET of the LSD-overexpressing cellfurther comprise detecting overexpression of a LSD gene (e.g., LSD1 orLSD2) (e.g., relative to the expression of the LSD gene in a normal cell(e.g., a normal breast cell)) in the LSD-overexpressing cell prior tocontacting the LSD-overexpressing cell with the LSD inhibitor. Innon-limiting examples of this type, the methods comprise detectingoverexpression of the LSD gene in a CSC. In other non-limiting examples,the methods comprise detecting overexpression of the LSD gene in anon-CSC tumor cell. In still other non-limiting examples, the methodscomprise detecting overexpression of the LSD gene in a CSC and a non-CSCtumor cell.

Suitably, the methods for altering at least one of: (i) formation; (ii)proliferation; (iii) survival; (iv) viability; (v) maintenance; (vi)EMT; or (vii) MET of the LSD-overexpressing cell further comprisedetecting that the LSD-overexpressing cell expresses one or more CSCmarkers as broadly described above.

In another aspect, the present invention provides methods for treatingor preventing a cancer (e.g., a metastatic cancer) in a subject, whereinthe cancer comprises at least one LSD-overexpressing cell (e.g., a LSD1or LSD2-overexpressing cell). These methods generally comprise, consistor consist essentially of administering to the subject a LSD inhibitorin an effective amount to alter at least one of: (i) formation; (ii)proliferation; (iii) survival; (iv) viability; (v) maintenance; (vi)EMT; or (vii) MET of the at least one LSD-overexpressing cell. Suitably,the LSD inhibitor is administered to the subject in an effective amountto inhibit (i) formation, (ii) proliferation, (iii) survival, (iv)viability, (v) maintenance, or (vi) EMT of the at least oneLSD-overexpressing cell, or to stimulate or induce (vii) MET of the atleast one LSD-overexpressing cell. In some embodiments, the LSDinhibitor is a selective LSD inhibitor. In other embodiments, the LSDinhibitor is a non-selective LSD inhibitor. Suitably, the at least oneLSD-overexpressing cell is selected from a CSC and a non-CSC tumor cell.

In some embodiments, the cancer is selected from breast, prostate, lung,bladder, pancreatic, colon, melanoma, liver or glioma cancer. Suitably,the cancer is breast cancer. In some embodiments, the CSCs give rise tonon-CSC tumor cells that are hormone-resistant.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting overexpression of a LSD gene in a tumorsample obtained from the subject, wherein the tumor sample comprises theat least one LSD-overexpressing cell (e.g., a CSC and/or a non-CSC tumorcell), prior to administering the LSD inhibitor to the subject.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting expression of one or more CSC markers asbroadly described above in a tumor sample obtained from the subject,wherein the tumor sample comprises the at least one LSD-overexpressingcell, prior to administering the LSD inhibitor to the subject.

Yet another aspect of the present invention provides methods fortreating or preventing a cancer (e.g., a metastatic cancer) in asubject, wherein the cancer comprises a CSC and a non-CSC tumor cell.These methods generally comprise, consist or consist essentially ofconcurrently administering to the subject (1) a LSD inhibitor in aneffective amount to inhibit at least one of: (i) formation, (ii)proliferation, (iii) survival, (iv) viability, or (v) maintenance of theCSC and/or the non-CSC tumor cell; and/or to inhibit (vi) EMT of theCSC; and/or to stimulate or induce (vii) MET of the CSC, and (2) acancer therapy or agent that inhibits the proliferation, survival orviability of the non-CSC tumor cell, to thereby treat or prevent thecancer. Suitably, the LSD inhibitor is administered to the subject in aneffective amount to inhibit (i) formation, (ii) proliferation, (iii)survival, (iv) viability, or (v) maintenance of the CSC and/or non-CSCtumor cell, and/or to inhibit (vi) EMT of the CSC, and/or to stimulateor induce (vii) MET of the CSC. In some embodiments, the LSD inhibitoris a selective LSD inhibitor. In other embodiments, the LSD inhibitor isa non-selective LSD inhibitor. In some embodiments, the cancer therapyor agent is selected from radiotherapy, surgery, chemotherapy, hormoneablation therapy, pro-apoptosis therapy and immunotherapy. Inillustrative examples of this type, the cancer therapy or agent targetsrapidly dividing cells or disrupts the cell cycle or cell division.

Suitably, the methods further comprise identifying that the subject hasor is at risk of developing a cancer comprising the CSC and the non-CSCtumor cell prior to the co-administration. In some embodiments, thecancer is selected from breast, prostate, lung, bladder, pancreatic,colon, melanoma, liver or glioma cancer. Suitably, the cancer isselected from breast, prostate, lung, bladder, pancreatic, colon,melanoma, liver or brain cancer.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting overexpression of a LSD gene (e.g., relativeto the expression of LSD in a normal cell (e.g., a normal breast cell))in a tumor sample obtained from the subject, wherein the tumor samplecomprises the CSC or the non-CSC tumor cell or both, prior toadministering the LSD inhibitor to the subject.

In some embodiments, the methods for treating or preventing a cancerfurther comprise detecting that the CSC expresses one or more CSCmarkers as broadly described above prior to administering the LSDinhibitor to the subject.

Suitably, the LSD inhibitor and the cancer therapy agent areadministered in synergistically effective amounts.

Typically, one or both of the LSD inhibitor and the cancer therapy oragent are administered on a routine schedule, for example, every day, atleast twice a week, at least three times a week, at least four times aweek, at least five times a week, at least six times a week, every week,every other week, every third week, every fourth week, every month,every two months, every three months, every four months, and every sixmonths.

In yet another aspect, the invention provides methods for identifyingagents that are useful for inhibiting (i) formation, (ii) proliferation,(iii) survival, (iv) viability, or (v) maintenance of a LSD (e.g., LSD1or LSD2)-overexpressing cell (e.g., a CSC and/or a non-CSC tumor cell),or for inhibiting (vi) EMT of a LSD-overexpressing cell (e.g., a CSC),or for stimulating or inducing (vii) MET of a LSD-overexpressing cell(e.g., a CSC), or for treating or preventing a cancer in a subject,wherein the cancer comprises a LSD-overexpressing cell (e.g., a CSCand/or a non-CSC tumor cell). These methods generally comprisecontacting a preparation with a test agent, wherein the preparationcomprises (i) a polypeptide comprising an amino acid sequencecorresponding to at least a biologically active fragment of a LSD, or toa variant or derivative thereof; or (ii) a polynucleotide comprising anucleotide sequence from which a transcript of a LSD gene or portionthereof is producible, or (iii) a polynucleotide comprising at least aportion of a genetic sequence (e.g., a transcriptional element) thatregulates the expression of a LSD gene, which is operably linked to areporter gene. A detected reduction in the level and/or functionalactivity (e.g., as broadly described above) of the polypeptide,transcript or transcript portion or an expression product of thereporter gene, relative to a normal or reference level and/or functionalactivity in the absence of the test agent, indicates that the agent isuseful for inhibiting (i) formation, (ii) proliferation, (iii) survival,(iv) viability, or (v) maintenance of a LSD-overexpressing cell (e.g., aCSC and/or a non-CSC tumor cell), or for inhibiting (vi) EMT of aLSD-overexpressing cell (e.g., a CSC), or for stimulating or inducing(vii) MET of a LSD-overexpressing cell (e.g., a CSC), or for treating orpreventing a cancer.

Still another aspect of the present invention provides methods ofproducing an agent for inhibiting at least one of: (i) formation, (ii)proliferation, (iii) survival, (iv) viability, or (v) maintenance of aLSD (e.g., LSD1 or LSD2)-overexpressing cell (e.g., a CSC and/or anon-CSC tumor cell), or for inhibiting (vi) EMT of a LSD-overexpressingcell (e.g., a CSC), or for stimulating or inducing (vii) MET of aLSD-overexpressing cell (e.g., a CSC), or for treating or preventing acancer in a subject, wherein the cancer comprises a LSD-overexpressingcell (e.g., a CSC and/or a non-CSC tumor cell), as broadly describedabove. These methods generally comprise: testing an agent suspected ofinhibiting a LSD as broadly described above; and synthesizing the agenton the basis that it tests positive for the inhibition. Suitably, themethod further comprises derivatizing the agent, and optionallyformulating the derivatized agent with a pharmaceutically acceptablecarrier and/or diluent, to improve the efficacy of the agent forinhibiting (i) formation, (ii) proliferation, (iii) survival, (iv)viability, or (v) maintenance of a LSD-overexpressing cell (e.g., a CSCand/or a non-CSC tumor cell), or for inhibiting (vi) EMT of aLSD-overexpressing cell (e.g., a CSC), or for stimulating or inducing(vii) MET of a LSD-overexpressing cell (e.g., a CSC), or for treating orpreventing a cancer in a subject, wherein the cancer comprises aLSD-overexpressing cell (e.g., a CSC and/or a non-CSC tumor cell).

Another aspect of the present invention provides pharmaceuticalcompositions for inhibiting at least one of: (i) formation, (ii)proliferation, (iii) survival, (iv) viability, or (v) maintenance of aCSC and/or a non-CSC tumor cell; and/or for inhibiting (vi) EMT of aCSC; and/or for stimulating (vii) MET of a CSC; and/or for treating orpreventing a cancer that comprises a CSC and/or a non-CSC tumor cell, asbroadly described above. These compositions generally comprise, consistor consist essentially of a LSD inhibitor and a second/auxiliary agentthat inhibits the proliferation, survival or viability of the non-CSCtumor cell. In some embodiments, the LSD inhibitor is a selective LSDinhibitor. In other embodiments, the LSD inhibitor is a non-selectiveLSD inhibitor.

In a further aspect, the present invention provides the use of a LSD(e.g., LSD1 or LSD2) inhibitor for altering at least one of: (i)formation; (ii) proliferation; (iii) survival; (iv) viability; (v)maintenance; (vi) EMT; or (vii) MET of a LSD-overexpressing cell or fortreating or preventing a cancer that comprises a LSD-overexpressing cell(e.g., a CSC or a non-CSC tumor cell), as broadly described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical and photographic representation showing in vitromeasurement of (A) key surface markers of human breast CSCs; (B)CSC-inducible genes in human breast CSCs; and (C) mammospheres in cellculture.

FIG. 2 is a graphical representation showing the results of H3K4me1 andLSD1 ChIP assays across the CD44 promoter region in three human CSCmodels. (EC, MCF-7 epithelial cell line; BC, MDA-MB 231 basal cellline).

FIG. 3 is a photographic representation showing nuclear staining of LSD1in human normal (A) and breast cancer tissue (B) and surface marker CD44in human normal (C) and breast cancer tissue (D)

FIG. 4 is a graphical representation showing that LSD1 knockdown bysiRNA inhibits the development of breast human CD44^(high) CD24^(low)CSCs in MDA MB-231 basal metastatic model: Transcript analysis (A), FACSanalysis (B), MCF-7 IM model: FACS analysis (C), Transcript analysis (D)and by ChIP (E) and FAIRE analysis (F).

FIG. 5 is a graphical representation showing that LSD2 knockdown bysiRNA inhibits the development of breast human CD44^(high) CD24^(low)CSCs using FACS analysis (A and B) and mammosphere assay (C).

FIG. 6 is a graphical and photographic representation showing that NCD38inhibits formation of CSCs in MCF-IM model. (A) LSD1 specific inhibitorNCD38 inhibits CD44 high CD24 low-CSC-like subpopulation in MCF-IMmodel. MCF-7 cells were either pre-incubated with vehicle alone or withNCD38 (5 μM for 17 hr), prior to PMA (0.65 ng/μl for 60 hours)stimulation (ST) or. Cells were subsequently stained with Hoechst 33528,APC-anti-CD44 and PE-anti-CD24 for 20 minutes on ice and subjected toFACS analysis. Circles on FACS plot indicate appropriate gating ofCD44^(high)/CD24^(low) CSC-like subpopulation and % CSC-likesubpopulation is shown above the gates respectively. (B) Graphicalrepresentation of data in FIG. 6A above. Data represent themean±standard error (SE) of three independent experiments. (C) 5 μMNCD38 reduce mammosphere formation in MCF-IM model. Mammosphere assaywas performed with 4×10⁴ MCF-7 cells/well in an ultra low attachment 6well plates. MCF-7 cells were pre-incubated either with vehicle alone orNCD38 (5 μM for 17 hr) prior to PMA stimulation (0.65 ng/ml for 6 days)(ST) or left non-stimulated (NS). Phase contrast microscopic images ofmammospheres were taken after 6 days of assay and only mammosphereslarger than 60 μm were counted. (D) Graphical representation of FIG. 6Cabove. Data represent the mean±standard error (SE) of three independentexperiments.

FIG. 7 is a graphical and photographic representation showing that thespecific LSD 1 inhibitor NCD38 inhibits maintenance of human metastaticbreast cancer cells and converts the cells to an epithelial form. (A)NCD38 inhibits CD44 high CD24 low-CSC-like subpopulation inBasal/metastatic model. MDA-MB 231 cells were either incubated withvehicle alone or with NCD38 (5 μM). Cells were subsequently stained withHoechst 33528, APC-anti-CD44 and PE-anti-CD24 for 20 minutes on ice andsubjected to FACS analysis. Percent CSC-like subpopulation is shown inthe bar graph. Data represent the mean±standard error (SE) of threeindependent experiments. (B) Graphical representation of FIG. 7A above.Data represent the mean±standard error (SE) of three independentexperiments. (C) LSD1 inhibitor NCD38 inhibit EMT in Basal/metastaticmodel. Phase contrast microscopy images of MDA-MB 231 cells werecaptured either without pre-treatment of LSD1 specific inhibitor(-Control) or with treatment of NCD38 (5 μM) (+NCD38).

FIG. 8 is a photographic representation showing that the LSD1 inhibitorpargyline in combination with chemotherapeutic agent docetaxel reducessize of tumor in in vivo mice xenograft model. 5×10⁶ MDA-MB-231 cellswere injected into the mammary fat pad of each mouse. Treatments wereinitiated at a tumor volume of 50 mm³ at the stated doses. (A)Representative mice from each treatment group after four weeks oftreatment. (B) Weekly tumor sizes of dissected tumors at week four oftreatment.

FIG. 9 is a graphical representation showing that pargyline incombination with docetaxel reduces volume and weight of tumor in in vivomice xenograft model after seven weeks of treatment initiation. 5×10⁶MDA-MB-231 cells were injected into the mammary fat pad of each mouse.Treatments were initiated at a tumor volume of 50 mm³ at the stateddoses. Tumor volumes were measured every week until seven weeks oftreatment. (A) Tumor weight in grams from each treatment group afterseven weeks of treatment. Tumor weight is mean±standard error (SE) ofthree mice. (B) Weekly tumor volumes in mm³. Days for chemotherapytreatments has been shown by black arrows on the graph. Tumor volume ismean±standard error (SE) of five or more mice.

FIG. 10 is a graphical representation showing that pargyline incombination with docetaxel reduces cancer stem cells of in vivo micetumors. 5×10⁶ MDA-MB-231 cells were injected into the mammary fat pad ofeach mouse. Treatments were initiated at a tumor volume of 50 mm³ at thestated doses. Mice were sacrificed on seven weeks of treatment. Singlecell suspensions were made and cells were subsequently stained withHoechst 33528, APC-anti-CD44 and PE-anti-CD24 for 20 minutes on ice andsubjected to FACS analysis. (A) Percent CSC subpopulation from eachtreatment group. (B) CD44 mRNA expression of dissected tumors from eachgroup by real-time PCR. Data represent the mean±standard error (SE) offive mice in each group.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “administration concurrently” or “administering concurrently”or “co-administering” and the like refer to the administration of asingle composition containing two or more actives, or the administrationof each active as separate compositions and/or delivered by separateroutes either contemporaneously or simultaneously or sequentially withina short enough period of time that the effective result is equivalent tothat obtained when all such actives are administered as a singlecomposition. By “simultaneously” is meant that the active agents areadministered at substantially the same time, and desirably together inthe same formulation. By “contemporaneously” it is meant that the activeagents are administered closely in time, e.g., one agent is administeredwithin from about one minute to within about one day before or afteranother. Any contemporaneous time is useful. However, it will often bethe case that when not administered simultaneously, the agents will beadministered within about one minute to within about eight hours andsuitably within less than about one to about four hours. Whenadministered contemporaneously, the agents are suitably administered atthe same site on the subject. The term “same site” includes the exactlocation, but can be within about 0.5 to about 15 centimeters,preferably from within about 0.5 to about 5 centimeters. The term“separately” as used herein means that the agents are administered at aninterval, for example at an interval of about a day to several weeks ormonths. The active agents may be administered in either order. The term“sequentially” as used herein means that the agents are administered insequence, for example at an interval or intervals of minutes, hours,days or weeks. If appropriate the active agents may be administered in aregular repeating cycle.

The term “agent” or “modulatory agent” includes a compound that inducesa desired pharmacological and/or physiological effect. The term alsoencompass pharmaceutically acceptable and pharmacologically activeingredients of those compounds specifically mentioned herein includingbut not limited to salts, esters, amides, prodrugs, active metabolites,analogs and the like. When the above term is used, then it is to beunderstood that this includes the active agent per se as well aspharmaceutically acceptable, pharmacologically active salts, esters,amides, prodrugs, metabolites, analogs, etc. The term “agent” is not tobe construed narrowly but extends to small molecules, proteinaceousmolecules such as peptides, polypeptides and proteins as well ascompositions comprising them and genetic molecules such as RNA, DNA andmimetics and chemical analogs thereof as well as cellular agents. Theterm “agent” includes a cell that is capable of producing and secretinga polypeptide referred to herein as well as a polynucleotide comprisinga nucleotide sequence that encodes that polypeptide. Thus, the term“agent” extends to nucleic acid constructs including vectors such asviral or non-viral vectors, expression vectors and plasmids forexpression in and secretion in a range of cells.

The term “altering” and grammatical equivalents as used herein inreference to the level of any substance and/or phenomenon refers to anincrease and/or decrease in the quantity of the substance and/orphenomenon, regardless of whether the quantity is determinedobjectively, and/or subjectively.

By “antigen-binding molecule” is meant a molecule that has bindingaffinity for a target antigen. It will be understood that this termextends to immunoglobulins, immunoglobulin fragments andnon-immunoglobulin derived protein frameworks that exhibitantigen-binding activity.

“Antigenic or immunogenic activity” refers to the ability of apolypeptide, fragment, variant or derivative according to the inventionto produce an antigenic or immunogenic response in an animal, suitably amammal, to which it is administered, wherein the response includes theproduction of elements which specifically bind the polypeptide orfragment thereof.

As used herein, the term “alkyl” refers to a straight chain, branched orcyclic saturated hydrocarbon group having 1 to 10 carbon atoms. Whereappropriate, the alkyl group may have a specified number of carbonatoms, for example, C₁₋₆alkyl which includes alkyl groups having 1, 2,3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examplesof suitable alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl,3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 5-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl,octyl, nonyl, decyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyland cycloheptyl.

As used herein, the term “alkenyl” refers to a straight-chain, branchedor cyclic hydrocarbon group having one or more double bonds betweencarbon atoms and having 2 to 10 carbon atoms. Where appropriate, thealkenyl group may have a specified number of carbon atoms. For example,C₂-C₆ as in “C₂-C₆alkenyl” includes groups having 2, 3, 4, 5 or 6 carbonatoms in a linear or branched arrangement. Examples of suitable alkenylgroups include, but are not limited to, ethenyl, propenyl, isopropenyl,butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl,heptenyl, octenyl, nonenyl, decenyl, cyclopentenyl, cyclohexenyl andcyclohexadienyl.

“Aralkyl” means alkyl as defined above which is substituted with an arylgroup as defined above, e.g., —CH₂phenyl, —(CH₂)₂phenyl, —(CH₂)₃phenyl,—H₂CH(CH₃)CH₂phenyl, and the like and derivatives thereof.

As used herein, “aromatic” or “aryl” is intended to mean any stablemonocyclic or bicyclic carbon ring of up to 7 atoms in each ring,wherein at least one ring is aromatic. Examples of such aryl elementsinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.

In certain instances, substituents may be defined with a range ofcarbons that includes zero, such as (C₀-C₆)alkylene-aryl. If aryl istaken to be phenyl, this definition would include phenyl itself as wellas, for example, —CH₂Ph, —CH₂CH₂Ph, CH(CH₃)CH₂CH(CH₃)Ph.

It will also be recognized that the compounds described herein maypossess asymmetric centers and are therefore capable of existing in morethan one stereoisomeric form. The invention thus also relates tocompounds in substantially pure isomeric form at one or more asymmetriccenters e.g., greater than about 90% ee, such as about 95% or 97% ee orgreater than 99% ee, as well as mixtures, including racemic mixtures,thereof. Such isomers may be naturally occurring or may be prepared byasymmetric synthesis, for example using chiral intermediates, or bychiral resolution.

As used herein, the term “binds specifically,” “specificallyimmuno-interactive” and the like when referring to an antigen-bindingmolecule refers to a binding reaction which is determinative of thepresence of an antigen in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antigen-binding molecules bind to a particularantigen and do not bind in a significant amount to other proteins orantigens present in the sample. Specific binding to an antigen undersuch conditions may require an antigen-binding molecule that is selectedfor its specificity for a particular antigen. For example,antigen-binding molecules can be raised to a selected protein antigen,which bind to that antigen but not to other proteins present in asample. A variety of immunoassay formats may be used to selectantigen-binding molecules specifically immuno-interactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmuno-interactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

The term “cancer stem cell” or CSC refers to a cell that hastumor-initiating and tumor-sustaining capacity, including the ability toextensively proliferate, form new tumors and maintain cancerdevelopment, i.e., cells with indefinite proliferative potential thatdrive the formation and growth of tumors. CSCs are biologically distinctfrom the bulk tumor cells and possess characteristics associated withstem cells, specifically the ability to self renew and to propagate andgive rise to all cell types found in a particular cancer sample. Theterm “cancer stem cell” or CSC includes both gene alteration in stemcells (SCs) and gene alteration in a cell which becomes a CSC. Inspecific embodiments, the CSCs breast CSCs, which are suitably CD24⁺CD44⁺, illustrative examples of which include CD44^(high) CD24^(low).

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. Thus, use of the term “comprising” and the likeindicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of”. Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

By “corresponds to” or “corresponding to” is meant a nucleic acidsequence that displays substantial sequence identity to a referencenucleic acid sequence (e.g., at least about 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to allor a portion of the reference nucleic acid sequence) or an amino acidsequence that displays substantial sequence similarity or identity to areference amino acid sequence (e.g., at least 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarityor identity to all or a portion of the reference amino acid sequence).

The term “derivatize,” “derivatizing” and the like refer to producing orobtaining a compound from another substance by chemical reaction, e.g.,by adding one or more reactive groups to the compound by reacting thecompound with a functional group-adding reagent, etc.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example by conjugation or complexingwith other chemical moieties or by post-translational modificationtechniques as would be understood in the art. The term “derivative” alsoincludes within its scope alterations that have been made to a parentsequence including additions or deletions that provide for functionalequivalent molecules.

The term “differentiation” of cancer stem cells as used herein refers toboth the change of cancer stem cells into pluripotent tumor progenitorsand the change of pluripotent tumor progenitors into unipotent tumorprogenitors and/or terminally differentiated tumor cells.

By “effective amount”, in the context of treating or preventing acondition is meant the administration of an amount of an agent orcomposition to an individual in need of such treatment or prophylaxis,either in a single dose or as part of a series, that is effective forthe prevention of incurring a symptom, holding in check such symptoms,and/or treating existing symptoms, of that condition. The effectiveamount will vary depending upon the health and physical condition of theindividual to be treated, the taxonomic group of individual to betreated, the formulation of the composition, the assessment of themedical situation, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials.

As used herein, the term “epithelial-to-mesenchymal transition” (EMT)refers to the conversion from an epithelial to a mesenchymal phenotype,which is a normal process of embryonic development. EMT is also theprocess whereby injured epithelial cells that function as ion and fluidtransporters become matrix remodeling mesenchymal cells. In carcinomas,this transformation typically results in altered cell morphology, theexpression of mesenchymal proteins and increased invasiveness. Thecriteria for defining EMT in vitro involve the loss of epithelial cellpolarity, the separation into individual cells and subsequent dispersionafter the acquisition of cell motility (see, Vincent-Salomon et al.,Breast Cancer Res. 2003; 5(2): 101-106). Classes of molecules thatchange in expression, distribution, and/or function during EMT, and thatare causally involved, include growth factors (e.g., transforming growthfactor (TGF)-β, wnts), transcription factors (e.g., Snail, SMAD, LEF,and nuclear β-catenin), molecules of the cell-to-cell adhesion axis(cadherins, catenins), cytoskeletal modulators (Rho family), andextracellular proteases (matrix metalloproteinases, plasminogenactivators) (see, Thompson et al., Cancer Research 65, 5991-5995, Jul.15, 2005).

As used herein, the term “epithelium” refers to the covering of internaland external surfaces of the body, including the lining of vessels andother small cavities. It consists of a collection of epithelial cellsforming a relatively thin sheet or layer due to the constituent cellsbeing mutually and extensively adherent laterally by cell-to-celljunctions. The layer is polarized and has apical and basal sides.Despite the tight regimentation of the epithelial cells the epitheliumdoes have some plasticity and cells in an epithelial layer can altershape, such as change from flat to columnar, or pinch in at one end andexpand at the other. However, these tend to occur in cell groups ratherthan individually (see, Thompson et al., 2005, supra).

The term “expression” refers the biosynthesis of a gene product. Forexample, in the case of a coding sequence, expression involvestranscription of the coding sequence into mRNA and translation of mRNAinto one or more polypeptides. Conversely, expression of a non-codingsequence involves transcription of the non-coding sequence into atranscript only.

By “expression vector” is meant any genetic element capable of directingthe transcription of a polynucleotide contained within the vector andsuitably the synthesis of a peptide or polypeptide encoded by thepolynucleotide. Such expression vectors are known to practitioners inthe art.

As used herein, the term “function” refers to a biological, enzymatic,or therapeutic function.

The term “gene” as used herein refers to any and all discrete codingregions of the cell's genome, as well as associated non-coding andregulatory regions. The term is intended to mean the open reading frameencoding specific polypeptides, introns, and adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression. In this regard, the gene may further comprise controlsignals such as promoters, enhancers, termination and/or polyadenylationsignals that are naturally associated with a given gene, or heterologouscontrol signals. The DNA sequences may be cDNA or genomic DNA or afragment thereof. The gene may be introduced into an appropriate vectorfor extrachromosomal maintenance or for integration into the host.

The term “group” as applied to chemical species refers to a set of atomsthat forms a portion of a molecule. In some instances, a group caninclude two or more atoms that are bonded to one another to form aportion of a molecule. A group can be monovalent or polyvalent (e.g.,bivalent) to allow bonding to one or more additional groups of amolecule. For example, a monovalent group can be envisioned as amolecule with one of its hydrogen atoms removed to allow bonding toanother group of a molecule. A group can be positively or negativelycharged. For example, a positively charged group can be envisioned as aneutral group with one or more protons (i.e., H⁺) added, and anegatively charged group can be envisioned as a neutral group with oneor more protons removed. Non-limiting examples of groups include, butare not limited to, alkyl groups, alkylene groups, alkenyl groups,alkenylene groups, alkynyl groups, alkynylene groups, aryl groups,arylene groups, iminyl groups, iminylene groups, hydride groups, halogroups, hydroxy groups, alkoxy groups, carboxy groups, thio groups,alkylthio groups, disulfide groups, cyano groups, nitro groups, aminogroups, alkylamino groups, dialkylamino groups, silyl groups, and siloxygroups. Groups such as alkyl, alkenyl, alkynyl, aryl, and heterocyclyl,whether used alone or in a compound word or in the definition of a groupmay be optionally substituted by one or more substituents. “Optionallysubstituted,” as used herein, refers to a group may or may not befurther substituted with one or more groups selected from alkyl,alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl,haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy,haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl,nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino,dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino,phenylamino, diphenylamino, benzylamino, dibenzylamino, hydrazino, acyl,acylamino, diacylamino, acyloxy, heterocyclyl, heterocycloxy,heterocyclamino, haloheterocyclyl, carboxy ester, carboxy, carboxyamide, mercapto, alkylthio, benzylthio, acylthio andphosphorus-containing groups. As used herein, the term “optionallysubstituted” may also refer to the replacement of a CH₂ group with acarbonyl (C═O) group. Non-limiting examples of optional substituentsinclude alkyl, preferably C₁₋₈ alkyl (e.g., C₁₋₆ alkyl such as methyl,ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl), hydroxy C₁₋₈ alkyl (e.g., hydroxymethyl, hydroxyethyl,hydroxypropyl), alkoxyalkyl (e.g., methoxymethyl, methoxyethyl,methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl etc.) C₁₋₈alkoxy, (e.g., C₁₋₆ alkoxy such as methoxy, ethoxy, propoxy, butoxy,cyclopropoxy, cyclobutoxy), halo (fluoro, chloro, bromo, iodo),monofluoromethyl, monochloromethyl, monobromomethyl, difluoromethyl,dichloromethyl, dibromomethyl, trifluoromethyl, trichloromethyl,tribromomethyl, hydroxy, phenyl (which itself may be furthersubstituted, by an optional substituent as described herein, e.g.,hydroxy, halo, methyl, ethyl, propyl, butyl, methoxy, ethoxy, acetoxy,amino), benzyl (wherein the CH₂ and/or phenyl group may be furthersubstituted as described herein), phenoxy (wherein the CH₂ and/or phenylgroup may be further substituted as described herein), benzyloxy(wherein the CH₂ and/or phenyl group may be further substituted asdescribed herein), amino, C₁₋₈ alkylamino (e.g., C₁₋₆ alkyl, such asmethylamino, ethylamino, propylamino), di C₁₋₈ alkylamino (e.g., C₁₋₆alkyl, such as dimethylamino, diethylamino, dipropylamino), acylamino(e.g., NHC(O)CH₃), phenylamino (wherein phenyl itself may be furthersubstituted as described herein), nitro, formyl, —C(O)—C₁₋₈ alkyl (e.g.,C₁₋₆ alkyl, such as acetyl), O—C(O)-alkyl (e.g., C₁₋₆ alkyl, such asacetyloxy), benzoyl (wherein the CH₂ and/or phenyl group itself may befurther substituted), replacement of CH₂ with C═O, CO₂H, CO₂ C₁₋₈ alkyl(e.g., C₁₋₆ alkyl such as methyl ester, ethyl ester, propyl ester, butylester), CO₂phenyl (wherein phenyl itself may be further substituted),CONH₂, CONHphenyl (wherein phenyl itself may be further substituted asdescribed herein), CONHbenzyl (wherein the CH₂ and/or phenyl group maybe further substituted as described herein), CONH C₁₋₈ alkyl (e.g., C₁₋₆alkyl such as methyl amide, ethyl amide, propyl amide, butyl amide),CONH C₁₋₈ alkylamine (e.g., C₁₋₆ alkyl such as aminomethyl amide,aminoethyl amide, aminopropyl amide, aminobutyl amide),—C(O)heterocyclyl (e.g., —C(O)-1-piperidine, —C(O)-1-piperazine,—C(O)-4-morpholine), —C(O)heteroaryl (e.g., —C(O)-1-pyridine,—C(O)-1-pyridazine, —C(O)-1-pyrimidine, —C(O)-1-pyrazine), CONHdi C₁₋₈alkyl (e.g., C₁₋₆alkyl).

“Heteroaralkyl” group means alkyl as defined above which is substitutedwith a heteroaryl group, e.g., —CH₂pyridinyl, —(CH₂)₂pyrimidinyl,—(CH₂)₃imidazolyl, and the like, and derivatives thereof.

The term “heteroaryl” or “heteroaromatic”, as used herein, represents astable monocyclic or bicyclic ring of up to 7 atoms in each ring,wherein at least one ring is aromatic and contains from 1 to 4heteroatoms selected from the group consisting of 0, N and S. Heteroarylgroups within the scope of this definition include but are not limitedto: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl,indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl,quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl,pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. Aswith the definition of heterocycle below, “heteroaryl” is alsounderstood to include the N-oxide derivative of any nitrogen-containingheteroaryl.

Further examples of “heterocyclyl” and “heteroaryl” include, but are notlimited to, the following: benzoimidazolyl, benzofuranyl,benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazoyl,indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof. Attachment of a heterocyclyl substituent can occur viaa carbon atom or via a heteroatom.

As used herein, “heteroarylene” refers to a bivalent monocyclic ormulticyclic ring system, preferably of about 3 to about 15 members whereone or more, more preferably 1 to 3 of the atoms in the ring system is aheteroatom, that is, an element other than carbon, for example,nitrogen, oxygen and sulfur atoms. The heteroarylene group may beoptionally substituted with one or more, preferably 1 to 3, aryl groupsubstituents. Exemplary heteroarylene groups include, for example,1,4-imidazolylene.

The term “heterocycle”, “heteroaliphatic” or “heterocyclyl” as usedherein is intended to mean a 5- to 10-membered nonaromatic heterocyclecontaining from 1 to 4 heteroatoms selected from the group consisting ofO, N and S, and includes bicyclic groups.

“Heterocyclylalkyl” group means alkyl as defined above which issubstituted with a heterocycle group, e.g., —CH₂pyrrolidin-1-yl,—(CH₂)₂piperidin-1-yl, and the like, and derivatives thereof.

The term “high,” as used herein, refers to a measure that is greaterthan normal, greater than a standard such as a predetermined measure ora subgroup measure or that is relatively greater than another subgroupmeasure. For example, CD44^(high) refers to a measure of CD44 that isgreater than a normal CD44 measure. Consequently, “CD44^(high)” alwayscorresponds to, at the least, detectable CD44 in a relevant part of asubject's body or a relevant sample from a subject's body. A normalmeasure may be determined according to any method available to oneskilled in the art. The term “high” may also refer to a measure that isequal to or greater than a predetermined measure, such as apredetermined cutoff. If a subject is not “high” for a particularmarker, it is “low” for that marker. In general, the cut-off used fordetermining whether a subject is “high” or “low” should be selected suchthat the division becomes clinically relevant.

“Homolog” is used herein to denote a gene or its product, which isrelated to another gene or product by decent from a common ancestral DNAsequence.

The term “hormone receptor negative (HR−) tumor” means a tumor that doesnot express a receptor for a hormone that stimulates the proliferation,survival or viability of the tumor above a certain threshold asdetermined by standard methods (e.g., immunohistochemical staining ofnuclei in the patients biological samples. The threshold may bemeasured, for example, using an Allred score or gene expression. See,e.g., Harvey et al. (1999. J Clin Oncol 17:1474-1481) and Badve et al.(2008. J Clin Oncol 26(15):2473-2481). In some embodiments, the tumordoes not express an estrogen receptor (ER−) and/or a progesteronereceptor (PR−).

The term “hormone receptor positive (HR+) tumor” means a tumor thatexpresses a receptor for a hormone that stimulates the proliferation,survival or viability of the tumor above a certain threshold asdetermined by standard methods (e.g., immunohistochemical staining ofnuclei in the patients biological samples. The threshold may bemeasured, for example, using an Allred score or gene expression. See,e.g., Harvey et al. (1999. J Clin Oncol 17:1474-1481) and Badve et al.(2008. J Clin Oncol 26(15):2473-2481). a tumor expressing eitherestrogen receptor (ER) or progesterone receptor (PR) as determined bystandard methods (e.g., immunohistochemical staining of nuclei in thepatients biological samples).

The term “hormone-resistant cancer” as used herein refers to a cancerthat has a decreased or eliminated response to a hormone therapy orendocrine therapy when compared to a non-hormone-resistant cancer. Froma biological and clinical standpoint, several patterns of resistance canbe distinguished: A) tumors that are inherently insensitive to endocrinereceptor (e.g., estrogen receptor) targeting despite endocrine receptorexpression (pan-endocrine therapy resistance or de novo resistance); B)tumors that are hormone dependent but resistant to one or more specificendocrine therapies (agent-selective resistance; for example respondedto tamoxifen but not aromatase inhibitor); and C) tumors that initiallyrespond to endocrine therapy but subsequently progress (acquiredresistance). All types of resistance are included herein. In someembodiments, the hormone-resistant cancer is a cancer that ishormone-resistant prior to the administration of a hormone or endocrinetherapy (i.e., it is de novo hormone-resistant). In other embodiments,the hormone-resistant cancer is a cancer that is initially nothormone-resistant, but becomes hormone-resistant after at least onetreatment of a hormone or endocrine therapy.

The term “hormone therapy” or “endocrine therapy” as used herein isdefined as a treatment pertaining to blocking or removing hormones. Thetreatment may remove the gland that synthesizes the hormone or theprohormone, block or inhibit hormone synthesis, or prevent or inhibitthe hormone from binding to its receptor, or down-regulate or degradethe hormone receptor.

“Hybridization” is used herein to denote the pairing of complementarynucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.Complementary base sequences are those sequences that are related by thebase-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA Upairs with A and C pairs with G. In this regard, the terms “match” and“mismatch” as used herein refer to the hybridization potential of pairednucleotides in complementary nucleic acid strands. Matched nucleotideshybridize efficiently, such as the classical A-T and G-C base pairmentioned above. Mismatches are other combinations of nucleotides thatdo not hybridize efficiently. In the present invention, the preferredmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances as known tothose of skill in the art.

The phrase “hybridizing specifically to” and the like refer to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “hydrocarbyl” as used herein includes any radical containingcarbon and hydrogen including saturated, unsaturated, aromatic, straightor branched chain or cyclic including polycyclic groups. Hydrocarbylincludes but is not limited to C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,C₃-C₁₀cycloalkyl, aryl such as phenyl and naphthyl, Ar (C₁-C₈)alkyl suchas benzyl, any of which may be optionally substituted.

Reference herein to “immuno-interactive” includes reference to anyinteraction, reaction, or other form of association between moleculesand in particular where one of the molecules is, or mimics, a componentof the immune system.

As used herein, the term “inhibitor” means an agent that decreases orinhibits the function or biological activity of a LSD polypeptide (e.g.,LSD1—also known as lysine-specific histone demethylase 1A; lysine(K)-specific demethylase 1 (KDM1); lysine (K)-specific demethylase 1A(KDM1A); BRAF35-HDAC complex protein BHC110; FAD-binding proteinBRAF35-HDAC complex, 110 kDa subunit; amine oxidase (flavin containing)domain 2 (AOF2); lysine-specific histone demethylase 1; RP1-184J9.1—andLSD2—also known as lysine-specific histone demethylase 1B (KDM1B); amineoxidase flavin-containing 1 (AOF1); amine oxidase (flavin-containing)domain 1; flavin-containing amine oxidase domain-containing protein 1;lysine-specific histone demethylase 2; or the expression of a LSD gene(e.g., LSD1—also known as KDM1A; AOF2; BHC110; KDM1—and LSD2—also knownas KDM1B; AOF1; bA204B7.3; C6orf193; dJ298J15.2).

The term “low,” as used herein, refers to a measure that is lower thannormal, lower than a standard such as a predetermined measure or asubgroup measure or that is relatively lower than another subgroupmeasure. For example, CD24^(low) refers to a measure of CD24 that islower than a normal CD24 measure. A normal measure may be determinedaccording to any method available to one skilled in the art. The term“low” may also refer to a measure that is equal to or lower than apredetermined measure, such as a predetermined cutoff.

The term “lower alkyl” refers to straight and branched chain alkylgroups having from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl,iso-propyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl,2-methylpentyl, and the like. In some embodiments, the lower alkyl groupis methyl or ethyl.

The term “lower alkoxy” refers to straight and branched chain alkoxygroups having from 1 to 6 carbon atoms, such as methoxy, ethoxy,n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,n-hexoxy, 2-methyl-pentoxy, and the like. Usually, the lower alkoxygroup is methoxy or ethoxy.

As used herein, the term “mesenchyme” refers to the part of theembryonic mesoderm, consisting of loosely packed, unspecialized cellsset in a gelatinous ground substance, from which connective tissue,bone, cartilage, and the circulatory and lymphatic systems develop.Mesenchyme is a collection of cells which form a relatively diffusetissue network. Mesenchyme is not a complete cellular layer and thecells typically have only points on their surface engaged in adhesion totheir neighbors. These adhesions may also involve cadherin associations(see, Thompson et al., 2005, supra).

As used herein, the term “mesenchymal-to-epithelial transition” (MET) isa reversible biological process that involves the transition frommotile, multipolar or spindle-shaped mesenchymal cells to planar arraysof polarized cells called epithelia. MET is the reverse process of EMT.METs occur in normal development, cancer metastasis, and inducedpluripotent stem cell reprogramming.

By “modulating” is meant increasing or decreasing, either directly orindirectly, the level or functional activity of a target molecule. Forexample, an agent may indirectly modulate the level/activity byinteracting with a molecule other than the target molecule. In thisregard, indirect modulation of a gene encoding a target polypeptideincludes within its scope modulation of the expression of a firstnucleic acid molecule, wherein an expression product of the firstnucleic acid molecule modulates the expression of a nucleic acidmolecule encoding the target polypeptide.

The term “oligonucleotide” as used herein refers to a polymer composedof a multiplicity of nucleotide residues (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analoguesthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogues thereof). Thus, while the term “oligonucleotide”typically refers to a nucleotide polymer in which the nucleotideresidues and linkages between them are naturally occurring, it will beunderstood that the term also includes within its scope variousanalogues including, but not restricted to, peptide nucleic acids(PNAs), phosphoramidates, phosphorothioates, methyl phosphonates,2-O-methyl ribonucleic acids, and the like. The exact size of themolecule can vary depending on the particular application. Anoligonucleotide is typically rather short in length, generally fromabout 10 to 30 nucleotide residues, but the term can refer to moleculesof any length, although the term “polynucleotide” or “nucleic acid” istypically used for large oligonucleotides.

The term “operably connected” or “operably linked” as used herein meansplacing a structural gene under the regulatory control of a regulatoryelement including but not limited to a promoter, which then controls thetranscription and optionally translation of the gene. In theconstruction of heterologous promoter/structural gene combinations, itis generally preferred to position the genetic sequence or promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that genetic sequence or promoter andthe gene it controls in its natural setting; i.e., the gene from whichthe genetic sequence or promoter is derived. As is known in the art,some variation in this distance can be accommodated without loss offunction. Similarly, the preferred positioning of a regulatory sequenceelement with respect to a heterologous gene to be placed under itscontrol is defined by the positioning of the element in its naturalsetting; i.e., the genes from which it is derived.

The terms “overexpress,” “overexpression,” or “overexpressed”interchangeably refer to a gene (e.g., a LSD gene) that is transcribedor translated at a detectably greater level, usually in a cancer cell,in comparison to a normal cell. Overexpression therefore refers to bothoverexpression of protein and RNA (due to increased transcription, posttranscriptional processing, translation, post translational processing,altered stability, and altered protein degradation), as well as localoverexpression due to altered protein traffic patterns (increasednuclear localization), and augmented functional activity, e.g., as in anincreased enzyme hydrolysis of substrate. Overexpression can also be by10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to anormal cell or comparison cell (e.g., a breast cell).

The terms “patient,” “subject,” “host” or “individual” usedinterchangeably herein, refer to any subject, particularly a vertebratesubject, and even more particularly a mammalian subject, for whomtherapy or prophylaxis is desired. Suitable vertebrate animals that fallwithin the scope of the invention include, but are not restricted to,any member of the subphylum Chordata including primates (e.g., humans,monkeys and apes, and includes species of monkeys such from the genusMacaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/orrhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well asmarmosets (species from the genus Callithrix), squirrel monkeys (speciesfrom the genus Saimiri) and tamarins (species from the genus Saguinus),as well as species of apes such as chimpanzees (Pan troglodytes)),rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits,hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g.,goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g.,dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks,geese, companion birds such as canaries, budgerigars etc.), marinemammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizardsetc.), and fish. In specific embodiments, the subject is a primate suchas a human. However, it will be understood that the aforementioned termsdo not imply that symptoms are present.

By “pharmaceutically acceptable carrier” is meant a pharmaceuticalvehicle comprised of a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to a subject alongwith the selected active agent without causing any or a substantialadverse reaction. Carriers may include excipients and other additivessuch as diluents, detergents, coloring agents, wetting or emulsifyingagents, pH buffering agents, preservatives, transfection agents and thelike.

Similarly, a “pharmacologically acceptable” salt, ester, amide, prodrugor derivative of a compound as provided herein is a salt, ester, amide,prodrug or derivative that this not biologically or otherwiseundesirable.

The terms “polynucleotide,” “genetic material,” “genetic forms,”“nucleic acids” and “nucleotide sequence” include RNA, cDNA, genomicDNA, synthetic forms and mixed polymers, both sense and antisensestrands, and may be chemically or biochemically modified or may containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those skilled in the art.

“Phenylalkyl” means alkyl as defined above which is substituted withphenyl, e.g., —CH₂phenyl, —(CH₂)₂phenyl, —(CH₂)₃phenyl,CH₃CH(CH₃)CH₂phenyl, and the like and derivatives thereof. Phenylalkylis a subset of the aralkyl group.

The terms “polynucleotide variant” and “variant” refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions as known in the art (seefor example Sambrook et al., Molecular Cloning. A Laboratory Manual”,Cold Spring Harbor Press, 1989). These terms also encompasspolynucleotides in which one or more nucleotides have been added ordeleted, or replaced with different nucleotides. In this regard, it iswell understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains abiological function or activity of the reference polynucleotide. Theterms “polynucleotide variant” and “variant” also include naturallyoccurring allelic variants.

The terms “polypeptide,” “proteinaceous molecule,” “peptide” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues and to variants and synthetic analogues of the same. Thus,these terms apply to amino acid polymers in which one or more amino acidresidues is a synthetic non-naturally-occurring amino acid, such as achemical analogue of a corresponding naturally-occurring amino acid, aswell as to naturally-occurring amino acid polymers. These terms do notexclude modifications, for example, glycosylations, acetylations,phosphorylations and the like. Soluble forms of the subjectproteinaceous molecules are particularly useful. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid including, for example, unnatural amino acids orpolypeptides with substituted linkages.

The term “polypeptide variant” refers to polypeptides in which one ormore amino acids have been replaced by different amino acids. It is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide (conservative substitutions) as describedhereinafter. These terms also encompass polypeptides in which one ormore amino acids have been added or deleted, or replaced with differentamino acids.

The term “pro-drug” is used in its broadest sense and encompasses thosederivatives that are converted in vivo to the compounds of theinvention. Such derivatives would readily occur to those skilled in theart, and include, for example, compounds where a free hydroxy group isconverted into an ester derivative.

As used herein, the terms “prevent,” “prevented,” or “preventing,” referto a prophylactic treatment which increases the resistance of a subjectto developing the disease or condition or, in other words, decreases thelikelihood that the subject will develop the disease or condition aswell as a treatment after the disease or condition has begun in order toreduce or eliminate it altogether or prevent it from becoming worse.These terms also include within their scope preventing the disease orcondition from occurring in a subject which may be predisposed to thedisease or condition but has not yet been diagnosed as having it.

As used herein, “racemate” refers to a mixture of enantiomers.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents when used in reference to the level of asubstance and/or phenomenon in a first sample relative to a secondsample, mean that the quantity of substance and/or phenomenon in thefirst sample is lower than in the second sample by any amount that isstatistically significant using any art-accepted statistical method ofanalysis. In one embodiment, the reduction may be determinedsubjectively, for example when a patient refers to their subjectiveperception of disease symptoms, such as pain, fatigue, etc. In anotherembodiment, the reduction may be determined objectively, for examplewhen the number of CSCs and/or non-CSC tumor cells in a sample from apatient is lower than in an earlier sample from the patient. In anotherembodiment, the quantity of substance and/or phenomenon in the firstsample is at least 10% lower than the quantity of the same substanceand/or phenomenon in a second sample. In another embodiment, thequantity of the substance and/or phenomenon in the first sample is atleast 25% lower than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 50%lower than the quantity of the same substance and/or phenomenon in asecond sample. In a further embodiment, the quantity of the substanceand/or phenomenon in the first sample is at least 75% lower than thequantity of the same substance and/or phenomenon in a second sample. Inyet another embodiment, the quantity of the substance and/or phenomenonin the first sample is at least 90% lower than the quantity of the samesubstance and/or phenomenon in a second sample. Alternatively, adifference may be expressed as an “n-fold” difference.

The terms “salts,” “derivatives” and “prodrugs” includes anypharmaceutically acceptable salt, ester, hydrate, or any other compoundwhich, upon administration to the recipient, is capable of providing(directly or indirectly) a compound of the invention, or an activemetabolite or residue thereof. Suitable pharmaceutically acceptablesalts include salts of pharmaceutically acceptable inorganic acids suchas hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamicand hydrobromic acids, or salts of pharmaceutically acceptable organicacids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic,benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic,stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic andvaleric acids. Base salts include, but are not limited to, those formedwith pharmaceutically acceptable cations, such as sodium, potassium,lithium, calcium, magnesium, ammonium and alkylammonium. Also, basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl and diethylsulfate; and others. However, it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of theinvention since these may be useful in the preparation ofpharmaceutically acceptable salts. The preparation of salts and prodrugsand derivatives can be carried out by methods known in the art. Forexample, metal salts can be prepared by reaction of a compound of theinvention with a metal hydroxide. An acid salt can be prepared byreacting an appropriate acid with a compound of the invention.

The term “selective” refers to compounds that inhibit or displayantagonism towards a LSD without displaying substantial inhibition orantagonism towards another LSD or another enzyme such as a monoamineoxidase (MAO) (e.g., MAO A or MAO B). Accordingly, a compound that isselective for LSD1 exhibits a LSD1 selectivity of greater than about2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-foldwith respect to inhibition or antagonism of another LSD (i.e., a LSDother than LSD1 such as LSD2) or of another enzyme (e.g., a MAO). Insome embodiments, selective compounds display at least 50-fold greaterinhibition or antagonism towards a specified LSD than towards anotherLSD or another enzyme (e.g., a MAO). In still other embodiments,selective compounds inhibit or display at least 100-fold greaterinhibition or antagonism towards a specified LSD than towards anotherLSD or another enzyme (e.g., a MAO). In still other embodiments,selective compounds display at least 500-fold greater inhibition orantagonism towards L a specified LSD than towards another LSD or anotherenzyme (e.g., a MAO). In still other embodiments, selective compoundsdisplay at least 1000-fold greater inhibition or antagonism towards aspecified LSD than towards another LSD or another enzyme (e.g., a MAO).

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by anappropriate method. For example, sequence identity analysis may becarried out using the DNASIS computer program (Version 2.5 for windows;available from Hitachi Software engineering Co., Ltd., South SanFrancisco, Calif., USA) using standard defaults as used in the referencemanual accompanying the software.

“Similarity” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions as defined in Table2.

TABLE 2 ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile, Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Similarity may be determined using sequence comparison programs such asGAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395). In thisway, sequences of a similar or substantially different length to thosecited herein might be compared by insertion of gaps into the alignment,such gaps being determined, for example, by the comparison algorithmused by GAP.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein a “small molecule” refers to a composition that has amolecular weight of less than 3 kilodaltons (kDa), and typically lessthan 1.5 kilodaltons, and suitably less than about 1 kilodalton. Smallmolecules may be nucleic acids, peptides, polypeptides, peptidomimetics,carbohydrates, lipids or other organic (carbon-containing) or inorganicmolecules. As those skilled in the art will appreciate, based on thepresent description, extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, may be screenedwith any of the assays of the invention to identify compounds thatmodulate a bioactivity. A “small organic molecule” is an organiccompound (or organic compound complexed with an inorganic compound(e.g., metal)) that has a molecular weight of less than 3 kilodaltons,less than 1.5 kilodaltons, or even less than about 1 kDa.

“Stringency” as used herein refers to the temperature and ionic strengthconditions, and presence or absence of certain organic solvents, duringhybridization. The higher the stringency, the higher will be theobserved degree of complementarity between sequences. “Stringentconditions” as used herein refers to temperature and ionic conditionsunder which only polynucleotides having a high proportion ofcomplementary bases, preferably having exact complementarity, willhybridize. The stringency required is nucleotide sequence dependent anddepends upon the various components present during hybridization, and isgreatly changed when nucleotide analogues are used. Generally, stringentconditions are selected to be about 10° C. to 20° C. less than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a target sequence hybridizes to a complementaryprobe. It will be understood that a polynucleotide will hybridize to atarget sequence under at least low stringency conditions, preferablyunder at least medium stringency conditions and more preferably underhigh stringency conditions. Reference herein to low stringencyconditions include and encompass from at least about 1% v/v to at leastabout 15% v/v formamide and from at least about 1 M to at least about 2M salt for hybridization at 42° C., and at least about 1 M to at leastabout 2 M salt for washing at 42° C. Low stringency conditions also mayinclude 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2),7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii)0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at roomtemperature. Medium stringency conditions include and encompass from atleast about 16% v/v to at least about 30% v/v formamide and from atleast about 0.5 M to at least about 0.9 M salt for hybridization at 42°C., and at least about 0.5 M to at least about 0.9 M salt for washing at42° C. Medium stringency conditions also may include 1% Bovine SerumAlbumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS forhybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mMEDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 42° C. Highstringency conditions include and encompass from at least about 31% v/vto at least about 50% v/v formamide and from at least about 0.01 M to atleast about 0.15 M salt for hybridization at 42° C., and at least about0.01 M to at least about 0.15 M salt for washing at 42° C. Highstringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4(pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS;or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washingat a temperature in excess of 65° C. One embodiment of high stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 65° C. One embodiment of veryhigh stringency conditions includes hybridizing 0.5 M sodium phosphate,7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at65° C. Other stringent conditions are well known in the art. A skilledaddressee will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see CURRENT PROTOCOLS IN MOLECULARBIOLOGY (supra) at pages 2.10.1 to 2.10.16 and MOLECULAR CLONING. ALABORATORY MANUAL (Sambrook, et al., eds.) (Cold Spring Harbor Press1989) at sections 1.101 to 1.104.

By “substantially complementary” it is meant that an oligonucleotide ora subsequence thereof is sufficiently complementary to hybridize with atarget sequence. Accordingly, the nucleotide sequence of theoligonucleotide or subsequence need not reflect the exact complementarysequence of the target sequence. In a preferred embodiment, theoligonucleotide contains no mismatches and with the target sequence.

As used herein, the term “synergistic” means that the therapeutic effectof a LSD inhibitor (e.g., a LSD1 or LSD2 inhibitor) when administered incombination with a cancer therapy or agent (or vice-versa) is greaterthan the predicted additive therapeutic effects of the LSD inhibitor andthe cancer therapy or agent when administered alone. The term“synergistically effective amount” as applied to a LSD inhibitor and acancer therapy agent refers to the amount of each component in acomposition (generally a pharmaceutical composition), which is effectivefor inhibiting the formation, proliferation, survival, viability ormaintenance of CSCs and non-CSC tumor cells, to thereby treat or preventthe cancer, and which produces an effect which does not intersect, in adose-response plot of the dose of LSD inhibitor versus a dose of thecancer therapy agent versus inhibiting the formation, proliferation,survival, viability or maintenance of CSCs and non-CSC tumor cells,either the dose LSD inhibitor axis or the dose cancer therapy agentaxis. The dose response curve used to determine synergy in the art isdescribed for example by Sande et al. (see, p. 1080-1105 in A. Goodmanet al., ed., the Pharmacological Basis of Therapeutics, MacMillanPublishing Co., Inc., New York (1980)). The optimum synergistic amountscan be determined, using a 95% confidence limit, by varying factors suchas dose level, schedule and response, and using a computer-generatedmodel that generates isobolograms from the dose response curves forvarious combinations of the LSD inhibitor and the cancer therapy agent.The highest inhibition of formation, proliferation, survival, viabilityor maintenance of CSCs and non-CSC tumor cells on the dose responsecurve correlates with the optimum dosage levels.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be therapeutic in terms of a partial or complete cure for adisease or condition (e.g., a hematologic malignancy) and/or adverseaffect attributable to the disease or condition. These terms also coverany treatment of a condition or disease in a mammal, particularly in ahuman, and include: (a) inhibiting the disease or condition, i.e.,arresting its development; or (b) relieving the disease or condition,i.e., causing regression of the disease or condition.

The term “tumor,” as used herein, refers to any neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. The terms “cancer” and “cancerous”refer to or describe the physiological condition in mammals that istypically characterized in part by unregulated cell growth. As usedherein, the term “cancer” refers to non-metastatic and metastaticcancers, including early stage and late stage cancers. The term“precancerous” refers to a condition or a growth that typically precedesor develops into a cancer. By “non-metastatic” is meant a cancer that isbenign or that remains at the primary site and has not penetrated intothe lymphatic or blood vessel system or to tissues other than theprimary site. Generally, a non-metastatic cancer is any cancer that is aStage 0, I, or II cancer, and occasionally a Stage III cancer. By “earlystage cancer” is meant a cancer that is not invasive or metastatic or isclassified as a Stage 0, I, or II cancer. The term “late stage cancer”generally refers to a Stage III or Stage IV cancer, but can also referto a Stage II cancer or a substage of a Stage II cancer. One skilled inthe art will appreciate that the classification of a Stage II cancer aseither an early stage cancer or a late stage cancer depends on theparticular type of cancer. Illustrative examples of cancer include, butare not limited to, breast cancer, prostate cancer, ovarian cancer,cervical cancer, pancreatic cancer, colorectal cancer, lung cancer,hepatocellular cancer, gastric cancer, liver cancer, bladder cancer,cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma,melanoma, brain cancer, non-small cell lung cancer, squamous cell cancerof the head and neck, endometrial cancer, multiple myeloma, rectalcancer, and esophageal cancer. In an exemplary embodiment, the cancer isbreast cancer.

The term “tumor sample” as used herein means a sample comprising tumormaterial obtained from a cancerous patient. The term encompassesclinical samples, for example tissue obtained by surgical resection andtissue obtained by biopsy, such as for example a core biopsy or a fineneedle biopsy. The term also encompasses samples comprising tumor cellsobtained from sites other than the primary tumor, e.g., circulatingtumor cells, as well as well as preserved tumor samples, such asformalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.The term encompasses cells that are the progeny of the patient's tumorcells, e.g., cell culture samples derived from primary tumor cells orcirculating tumor cells. The term encompasses samples that may compriseprotein or nucleic acid material shed from tumor cells in vivo, e.g.,bone marrow, blood, plasma, serum, and the like. The term alsoencompasses samples that have been enriched for tumor cells or otherwisemanipulated after their procurement and samples comprisingpolynucleotides and/or polypeptides that are obtained from a patient'stumor material.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast orvirus, into which a polynucleotide can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector can be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector can contain any means for assuring self-replication.Alternatively, the vector can be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. A vector system cancomprise a single vector or plasmid, two or more vectors or plasmids,which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon. The choice of the vector will typicallydepend on the compatibility of the vector with the host cell into whichthe vector is to be introduced. In the present case, the vector ispreferably a viral or viral-derived vector, which is operably functionalin animal and preferably mammalian cells. Such vector may be derivedfrom a poxvirus, an adenovirus or yeast. The vector can also include aselection marker such as an antibiotic resistance gene that can be usedfor selection of suitable transformants. Examples of such resistancegenes are known to those of skill in the art and include the nptII genethat confers resistance to the antibiotics kanamycin and G418(Geneticin®) and the hph gene, which confers resistance to theantibiotic hygromycin B.

As used herein, underscoring or italicizing the name of a gene shallindicate the gene, in contrast to its protein product, which isindicated by the name of the gene in the absence of any underscoring oritalicizing. For example, “LSD1” shall mean the LSD1 gene, whereas“LSD1” shall indicate the protein product or products generated fromtranscription and translation and/or alternative splicing of the “LSD1”gene.

Each embodiment described herein is to be applied mutatis mutandis toeach and every embodiment unless specifically stated otherwise.

2. Compositions and Methods for Reducing or Abrogating the Formation,Proliferation or Viability of Cancer Stem Cells

The present invention is based in part on the determination that breastcancers, including hormone resistant breast cancers, are enriched forCSCs and that LSDs (e.g., LSD1 and LSD2) are overexpressed in those CSCsand in non-CSC tumor cells. Based on these findings, the presentinventors treated breast CSCs with LSD (e.g., LSD1 and LSD2) inhibitorsand found that they specifically stimulated CSC death as well as MET ofCSCs. Without wishing to be bound by any theory or mode of operation, itis proposed that LSDs, including LSD1 and LSD2, play a critical role intranscription of CSC-specific genes by deregulating active chromatindomains across their regulatory regions and that this deregulationstimulates not only the production of breast CSCs but also theproduction of CSCs generally.

Based on the above observations, the present inventors propose that LSD(e.g., LSD1 and LSD2) inhibition will result in reduced proliferation,survival or viability of CSCs and/or non-CSC tumor cells, and/or inreduced EMT of CSC, and/or in increased MET of CSC, which will in turnresult in fewer non-CSC tumor cells differentiating therefrom and inmore effective treatment of non-CSC tumor cells with a cancer therapy oragent.

Thus, in accordance with the present invention, methods and compositionsare provided that take advantage of a LSD inhibitor (e.g., a LSD1 orLSD2 inhibitor) to reduce or abrogate proliferation, survival orviability of CSCs and non-CSC tumor cells, and/or to reduce or abrogateEMT of CSC, and/or to stimulate or induce MET of CSC for the treatmentor prophylaxis of a cancer (e.g., a metastatic cancer). In specificembodiments, the LSD inhibitor (e.g., a LSD1 or LSD2 inhibitor) is usedin combination with a cancer therapy or agent that reduces theproliferation, survival or viability of non-CSC tumor cell progeny ofthose cells. The methods and compositions of the present invention arethus particularly useful in the treatment or prophylaxis of cancers,including metastatic cancers, as described hereafter.

2.1 LSD Inhibitors

The LSD inhibitor includes and encompasses any active agent that reducesthe accumulation, function or stability of a LSD; or decrease expressionof a LSD gene, and such inhibitors include without limitation, smallmolecules and macromolecules such as nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, polysaccharides,lipopolysaccharides, lipids or other organic (carbon containing) orinorganic molecules.

In some embodiments, the LSD inhibitor is an antagonistic nucleic acidmolecule that functions to inhibit the transcription or translation ofLSD (e.g., LSD1 or LSD2) transcripts. Representative transcripts of thistype include nucleotide sequences corresponding to any one the followingsequences: (1) human LSD1 nucleotide sequences as set forth for examplein GenBank Accession Nos. NM_015013.3, NP_001009999.1, andNM_001009999.2; human LSD2 nucleotide sequences as set forth for examplein GenBank Accession No. NM_153042.3; (2) nucleotide sequences thatshare at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequenceidentity with any one of the sequences referred to in (1); (3)nucleotide sequences that hybridize under at least low, medium or highstringency conditions to the sequences referred to in (1); (4)nucleotide sequences that encode any one of the following amino acidsequences: human LSD1 amino acid sequences as set forth for example inGenPept Accession Nos. NP_055828.2, NP_001009999.1 and 060341.2; humanLSD2 amino acid sequences as set forth for example in GenPept AccessionNos. NP_694587.3; (5) nucleotide sequences that encode an amino acidsequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99% sequence similarity with any one of the sequences referred to in(4); and nucleotide sequences that encode an amino acid sequence thatshares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequenceidentity with any one of the sequences referred to in (4).

Illustrative antagonist nucleic acid molecules include antisensemolecules, aptamers, ribozymes and triplex forming molecules, RNAi andexternal guide sequences. The nucleic acid molecules can act aseffectors, inhibitors, modulators, and stimulators of a specificactivity possessed by a target molecule, or the functional nucleic acidmolecules can possess a de novo activity independent of any othermolecules.

Antagonist nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, antagonistnucleic acid molecules can interact with LSD (e.g., LSD1 or LSD2) mRNAor the genomic DNA of LSD (e.g., LSD1 or LSD2) or they can interact witha LSD polypeptide e.g., LSD1 or LSD2). Often antagonist nucleic acidmolecules are designed to interact with other nucleic acids based onsequence homology between the target molecule and the antagonist nucleicacid molecule. In other situations, the specific recognition between theantagonist nucleic acid molecule and the target molecule is not based onsequence homology between the antagonist nucleic acid molecule and thetarget molecule, but rather is based on the formation of tertiarystructure that allows specific recognition to take place.

In some embodiments, anti-sense RNA or DNA molecules are used todirectly block the translation of LSD (e.g., LSD1 or LSD2) by binding totargeted mRNA and preventing protein translation. Antisense moleculesare designed to interact with a target nucleic acid molecule througheither canonical or non-canonical base pairing. The interaction of theantisense molecule and the target molecule may be designed to promotethe destruction of the target molecule through, for example, RNAseHmediated RNA-DNA hybrid degradation. Alternatively the antisensemolecule may be designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Non-limiting methods include in vitro selectionexperiments and DNA modification studies using DMS and DEPC. In specificexamples, the antisense molecules bind the target molecule with adissociation constant (K_(d)) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹°,or 10⁻¹². In specific embodiments, antisense oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between −10 and +10regions are employed.

Aptamers are molecules that interact with a target molecule, suitably ina specific way. Aptamers are generally small nucleic acids ranging from15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP and theophiline, as well as large molecules, suchas reverse transcriptase and thrombin. Aptamers can bind very tightlywith Kds from the target molecule of less than 10⁻¹² M. Suitably, theaptamers bind the target molecule with a Kd less than 10⁻⁶, 10⁻⁸, 10⁻¹°,or 10⁻¹². Aptamers can bind the target molecule with a very high degreeof specificity. For example, aptamers have been isolated that havegreater than a 10,000 fold difference in binding affinities between thetarget molecule and another molecule that differ at only a singleposition on the molecule. It is desirable that an aptamer have a K_(d)with the target molecule at least 10-, 100-, 1000-, 10,000-, or100,000-fold lower than the K_(d) with a background-binding molecule. Asuitable method for generating an aptamer to a target of interest (e.g.,PHD, FIH-1 or vHL) is the “Systematic Evolution of Ligands byEXponential Enrichment” (SELEX™). The SELEX™ method is described in U.S.Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163 (see also WO 91/19813).Briefly, a mixture of nucleic acids is contacted with the targetmolecule under conditions favorable for binding. The unbound nucleicacids are partitioned from the bound nucleic acids, and the nucleicacid-target complexes are dissociated. Then the dissociated nucleicacids are amplified to yield a ligand-enriched mixture of nucleic acids,which is subjected to repeated cycles of binding, partitioning,dissociating and amplifying as desired to yield highly specific highaffinity nucleic acid ligands to the target molecule.

In other embodiments, anti-LSD (e.g., anti-LSD1 or LSD2) ribozymes areused for catalyzing the specific cleavage of LSD (e.g., LSD1 or LSD2)RNA. The mechanism of ribozyme action involves sequence specifichybridization of the ribozyme molecule to complementary target RNA,followed by a endonucleolytic cleavage. There are several differenttypes of ribozymes that catalyze nuclease or nucleic acid polymerasetype reactions, which are based on ribozymes found in natural systems,such as hammerhead ribozymes, hairpin ribozymes, and tetrahymenaribozymes. There are also a number of ribozymes that are not found innatural systems, but which have been engineered to catalyze specificreactions de novo. Representative ribozymes cleave RNA or DNAsubstrates. In some embodiments, ribozymes that cleave RNA substratesare employed. Specific ribozyme cleavage sites within potential RNAtargets are initially identified by scanning the target molecule forribozyme cleavage sites, which include the following sequences, GUA, GUUand GUC. Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. The suitability of candidate targets may also beevaluated by testing their accessibility to hybridization withcomplementary oligonucleotides, using ribonuclease protection assays.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependent on both Watson-Crick and Hoogsteen base pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is generally desirable that thetriplex forming molecules bind the target molecule with a K_(d) lessthan 10⁻⁶, 10⁻⁸, 10⁻¹°, or 10⁻¹².

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNAseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. Similarly,eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized tocleave desired targets within eukaryotic cells.

In other embodiments, RNA molecules that mediate RNA interference (RNAi)of a LSD (e.g., LSD1 or LSD2) gene or LSD (e.g., LSD1 or LSD2)transcript can be used to reduce or abrogate gene expression. RNAirefers to interference with or destruction of the product of a targetgene by introducing a single-stranded or usually a double-stranded RNA(dsRNA) that is homologous to the transcript of a target gene. RNAimethods, including double-stranded RNA interference (dsRNAi) or smallinterfering RNA (siRNA), have been extensively documented in a number oforganisms, including mammalian cells and the nematode C. elegans (Fireet al., 1998. Nature 391, 806-811). In mammalian cells, RNAi can betriggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA(siRNA) (Chiu et al., 2002 Mol. Cell. 10:549-561; Elbashir et al., 2001.Nature 411:494-498), or by micro-RNAs (miRNA), functional small-hairpinRNA (shRNA), or other dsRNAs which are expressed in vivo using DNAtemplates with RNA polymerase III promoters (Zeng et al., 2002. Mol.Cell 9:1327-1333; Paddison et al., 2002. Genes Dev. 16:948-958; Lee etal., 2002. Nature Biotechnol. 20:500-505; Paul et al., 2002. NatureBiotechnol. 20:505-508; Tuschl, T., 2002. Nature Biotechnol. 20:440-448;Yu et al., 2002. Proc. Natl. Acad. Sci. USA 99(9):6047-6052; McManus etal., 2002. RNA 8:842-850; Sui et al., 2002. Proc. Natl. Acad. Sci. USA99(6):5515-5520).

In specific embodiments, dsRNA per se and especially dsRNA-producingconstructs corresponding to at least a portion of a LSD (e.g., LSD1 orLSD2) gene are used to reduce or abrogate its expression. RNAi-mediatedinhibition of gene expression may be accomplished using any of thetechniques reported in the art, for instance by transfecting a nucleicacid construct encoding a stem-loop or hairpin RNA structure into thegenome of the target cell, or by expressing a transfected nucleic acidconstruct having homology for a LSD (e.g., LSD1 or LSD2) gene frombetween convergent promoters, or as a head to head or tail to tailduplication from behind a single promoter. Any similar construct may beused so long as it produces a single RNA having the ability to fold backon itself and produce a dsRNA, or so long as it produces two separateRNA transcripts, which then anneal to form a dsRNA having homology to atarget gene.

Absolute homology is not required for RNAi, with a lower threshold beingdescribed at about 85% homology for a dsRNA of about 200 base pairs(Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev. 10:562-67). Therefore, depending on the length of the dsRNA, theRNAi-encoding nucleic acids can vary in the level of homology theycontain toward the target gene transcript, i.e., with dsRNAs of 100 to200 base pairs having at least about 85% homology with the target gene,and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about75% homology to the target gene. RNA-encoding constructs that express asingle RNA transcript designed to anneal to a separately expressed RNA,or single constructs expressing separate transcripts from convergentpromoters, are suitably at least about 100 nucleotides in length.RNA-encoding constructs that express a single RNA designed to form adsRNA via internal folding are usually at least about 200 nucleotides inlength.

The promoter used to express the dsRNA-forming construct may be any typeof promoter if the resulting dsRNA is specific for a gene product in thecell lineage targeted for destruction. Alternatively, the promoter maybe lineage specific in that it is only expressed in cells of aparticular development lineage. This might be advantageous where someoverlap in homology is observed with a gene that is expressed in anon-targeted cell lineage. The promoter may also be inducible byexternally controlled factors, or by intracellular environmentalfactors.

In some embodiments, RNA molecules of about 21 to about 23 nucleotides,which direct cleavage of specific mRNA to which they correspond, as forexample described by Tuschl et al. in U.S. 2002/0086356, can be utilizedfor mediating RNAi. Such 21- to 23-nt RNA molecules can comprise a 3′hydroxyl group, can be single-stranded or double stranded (as two 21- to23-nt RNAs) wherein the dsRNA molecules can be blunt ended or compriseoverhanging ends (e.g., 5′, 3′).

In some embodiments, the antagonist nucleic acid molecule is a siRNA.siRNAs can be prepared by any suitable method. For example, referencemay be made to International Publication WO 02/44321, which disclosessiRNAs capable of sequence-specific degradation of target mRNAs whenbase-paired with 3′ overhanging ends, which is incorporated by referenceherein. Sequence specific gene silencing can be achieved in mammaliancells using synthetic, short double-stranded RNAs that mimic the siRNAsproduced by the enzyme dicer. siRNA can be chemically or invitro-synthesized or can be the result of short double-strandedhairpin-like RNAs (shRNAs) that are processed into siRNAs inside thecell. Synthetic siRNAs are generally designed using algorithms and aconventional DNA/RNA synthesizer. Suppliers include Ambion (Austin,Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), GlenResearch (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can also besynthesized in vitro using kits such as Ambion's SILENCER™ siRNAConstruction Kit.

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAs (shRNAs). Kits for the productionof vectors comprising shRNA are available, such as, for example,Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™inducible RNAi plasmid and lentivirus vectors. In addition, methods forformulation and delivery of siRNAs to a subject are also well known inthe art. See, e.g., US 2005/0282188; US 2005/0239731; US 2005/0234232;US 2005/0176018; US 2005/0059817; US 2005/0020525; US 2004/0192626; US2003/0073640; US 2002/0150936; US 2002/0142980; and US2002/0120129, eachof which is incorporated herein by reference.

Illustrative RNAi molecules (e.g., LSD (e.g., LSD1 or LSD2) siRNA andshRNA) are described in the art (e.g., Yang, et al., 2010. Proc. Natl.Acad. Sci. USA 107: 21499-21504 and He et al., 2012. Transcription3:3:1-16) or available commercially from Santa Cruz Biotechnology, Inc.(Santa Cruz, Calif., USA) and OriGene Technologies, Inc. (Rockville,Md., USA).

The present invention further contemplates peptide or polypeptide basedinhibitor compounds. For example, BHC80 (also known as PHD fingerprotein 21A) forms part of a complex with LSD1 and can inhibit LSD1demethylase activity. Accordingly, the present invention furthercontemplates the use of BHC80 or biologically active fragments thereoffor inhibiting LSD1 enzymatic activity. Amino acid sequences of BHC80polypeptides, and nucleotide sequences encoding BHC80 polypeptides, arepublicly available. In this regard, reference may be made for example toGenBank Accession No. NP057705 for a Homo sapiens BHC80 amino acidsequence; and GenBank NM016621 for a nucleotide sequence encoding theamino acid sequence set forth in GenBank Accession No. NP057705; 2)GenBank Accession No. NP620094 for a Mus musculus BHC80 amino acidsequence; and GenBank NM138755 for a nucleotide sequence encoding theamino acid sequence set forth in GenBank Accession No. NP620094; 3)GenBank Accession No. NP00118576.1 for a Gallus gallus BHC80 amino acidsequence; and GenBank NM001199647 for a nucleotide sequence encoding theamino acid sequence set forth in GenBank Accession No. NP00118576.1; and4) GenBank Accession No. DAA21793 for a Bos taurus BHC80 amino acidsequence.

Illustrative BHC80 polypeptides are selected from the group consistingof: (1) a polypeptide comprising an amino acid sequence that shares atleast 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequencesimilarity with the amino acid sequence listed in any one of the GenBankBHC80 polypeptide entries noted above; (2) a polypeptide comprising anamino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99% sequence identity with the amino acid sequence listed inany one of the GenBank BHC80 polypeptide entries noted above; (3) apolypeptide comprising an amino acid sequence that is encoded by anucleotide sequence that hybridizes under at least low, medium or highstringency conditions to the nucleotide sequence listed in any one ofthe GenBank BHC80 polynucleotide entries noted above; (4) a polypeptidecomprising an amino acid sequence that is encoded by a nucleotidesequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99% sequence identity to the nucleotide sequence listed in any oneof the GenBank BHC80 polynucleotide entries noted above; and (5) afragment of a polypeptide according to any one of (1) to (4), whichinhibits LSD1 enzymatic activity.

A BHC80 polypeptide can be introduced into a cell by delivering apolypeptide per se, or by introducing into the cell a BHC80 nucleic acidcomprising a nucleotide sequence encoding a BHC80 polypeptide. In someembodiments, a BHC80 nucleic acid comprises a nucleotide sequenceselected from: (1) a BHC80 nucleotide sequence listed in any one of theGenBank BHC80 polynucleotide entries noted above; (2) a nucleotidesequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99% sequence identity with any one of the sequences referred to in(1); (3) a nucleotide sequence that hybridizes under at least low,medium or high stringency conditions to the sequences referred to in(1); (4) a nucleotide sequence that encodes an amino acid sequencelisted in any one of the GenBank BHC80 polypeptide entries noted above;(5) a nucleotide sequence that encodes an amino acid sequence thatshares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequencesimilarity with any one of the sequences referred to in (4); and anucleotide sequence that encodes an amino acid sequence that shares atleast 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequenceidentity with any one of the sequences referred to in (4).

The BHC80 nucleic acid can be in the form of a recombinant expressionvector. The BHC80 nucleotide sequence can be operably linked to atranscriptional control element(s), e.g., a promoter, in the expressionvector. Suitable vectors include, e.g., recombinant retroviruses,lentiviruses, and adenoviruses; retroviral expression vectors,lentiviral expression vectors, nucleic acid expression vectors, andplasmid expression vectors. In some cases, the expression vector isintegrated into the genome of a cell. In other cases, the expressionvector persists in an episomal state in a cell.

Suitable expression vectors include, but are not limited to, viralvectors (e.g., viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol V is Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson,PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999;WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol V is Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet. 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

The present invention also contemplates small molecule agents thatreduce enzymatic activity of LSDs (e.g., LSD1 or LSD2).

Small molecule agents that reduce enzymatic activity of LSD1 that aresuitable for use in the present invention include monoamine oxidase(MAO) inhibitors that also inhibit LSD1 enzymatic activity; polyaminecompounds that inhibit LSD1 enzymatic activity; phenylcyclopropylaminederivatives that inhibit LSD1 enzymatic activity; and the like.

Non-limiting examples of MAO inhibitors include MAO-A-selectiveinhibitors, MAO-B-selective inhibitors, and MAO non-selectiveinhibitors. Illustrative examples of MAO inhibitors include reportedinhibitors of the MAO-A isoform, which preferentially deaminates5-hydroxytryptamine (serotonin) (5-HT) and norepinephrine (NE), and/orthe MAO-B isoform, which preferentially deaminates phenylethylamine(PEA) and benzylamine (both MAO-A and MAO-B metabolize Dopamine (DA)).In various embodiments, MAO inhibitors may be irreversible or reversible(e.g., reversible inhibitors of MAO-A (RIMA)), and may have varyingpotencies against MAO-A and/or MAO-B (e.g., non-selective dualinhibitors or isoform-selective inhibitors).

In some embodiments, the MAO inhibitors are selected from: clorgyline;L-deprenyl; isocarboxazid (Marplan™); ayahuasca; nialamide; iproniazide;iproclozide; moclobemide (Aurorix™;4-chloro-N-(2-morpholin-4-ylethyl)benzamide); phenelzine (Nardil™;(±)-2-phenylethylhydrazine); tranylcypromine (Parnate™;(±)-trans-2-phenylcyclopropan-1-amine) (the congeneric of phenelzine);toloxatone; levo-deprenyl (Selegiline™); harmala; RIMAs (e.g.,moclobemide, described in Da Prada et al. (1989. J Pharmacol Exp Ther248:400-414); brofaromine; and befloxatone, described in Curet et al.(1998. J Affect Disord 51: 287-30), lazabemide (Ro 19 6327), describedin Ann. Neurol., 40(1): 99-107 (1996), and SL25.1131, described in Aubinet al. (2004. J. Pharmacol. Exp. Ther. 310: 1171-1182); selegilinehydrochloride (1-deprenyl, ELDEPRYL, ZELAPAR); dimethylselegilene;safinamide; rasagiline (AZILECT); bifemelane; desoxypeganine; harmine(also known as telepathine or banasterine); linezolid (ZYVOX, ZYVOXID);pargyline (EUDATIN, SUPIRDYL); dienolide kavapyrone desmethoxyyangonin;5-(4-Arylmethoxyphenyl)-2-(2-cyanoethyl)tetrazoles; and the like.

Small molecule LSD1 inhibitors may also be selected from polyaminecompounds as described for example by Woster et al. in U.S. PublicationNo. 2007/0208082, which is incorporated herein by reference in itsentirety. Illustrative polyamine inhibitors of LSD1 include compoundsaccording to formula (I):

or a salt, solvate, or hydrate thereof, where n is an integer from 1 to12; m and p are independently an integer from 1 to 5; q is 0 or 1; eachR₁ is independently selected from the group consisting of C₁-C₈ alkyl,C₄-C₁₅ cycloalkyl, C₃-C₁₅ branched alkyl, C₆-C₂₀ aryl, C₆-C₂₀heteroaryl, C₇-C₂₄ aralkyl, C₇-C₂₄ heteroaralkyl, and

where R₃ is selected from the group consisting of C₁-C₈ alkyl, C₄-C₁₅cycloalkyl, C₃-C₁₅ branched alkyl, C₆-C₂₀ aryl, C₆-C₂₀ heteroaryl,C₇-C₂₄ aralkyl and C₇-C₂₄ heteroaralkyl; and

each R₂ is independently selected from hydrogen or a C₁-C₈ alkyl.

A suitable polyamine compound is a compound of Formula (I), wherein oneor both R₁ is a C₆-C₂₀ aryl, such as a single ring aryl, includingwithout limitation, a phenyl. In one embodiment, the compound is of theformula (I) and each R₁ is phenyl. In one embodiment, q is I, m and pare 3, and n is 4. In another embodiment, q is I, m and p are 3, and nis 7.

A suitable polyamine compound is a compound of Formula (I), where atleast one or both R₁ is a C₈-C₁₂ or a C₁-C₈ alkyl, such as a linearalkyl. One or both R₁ may be a C₁-C₈ linear alkyl, such as methyl orethyl. In one embodiment, each R₁ is methyl. One or both R₁ may compriseor be a C₄-C₁₅ cycloalkyl group, such as a cycloalkyl group containing alinear alkyl group, where the cycloalkyl group is connected to themolecule either via its alkyl or cycloalkyl moiety. For instance, one orboth R₁ may be cyclopropylmethyl or cyclohexylmethyl. In one embodiment,one R₁ is cyclopropylmethyl or cyclohexylmethyl and the other R₁ is alinear alkyl group, such as a linear C₁-C₈ unsubstituted alkyl group,including without limitation an ethyl group. In one embodiment, R₁ is aC₃-C₁₅ branched alkyl group such as isopropyl. When R₁ is a C₁-C₈substituted alkyl, the substituted alkyl may be substituted with anysubstituent, including a primary, secondary, tertiary or quaternaryamine. Accordingly, in one embodiment, R₁ is a C₁-C₈ alkyl groupsubstituted with an amine such that R₁ may be e.g., alkyl-NH₂ or analkyl-amine-alkyl moiety such as —(CH₂)_(y)NH(CH₂)_(z)CH₃ where y and zare independently an integer from 1 to 8. In one embodiment, R₁ is—(CH₂)₃NH₂.

In one embodiment, the compound is of the formula (I) where one or bothR₁ is a C₇-C₂₄ substituted or unsubstituted aralkyl, which in oneembodiment is an aralkyl connected to the molecule via its alkyl moiety(e.g., benzyl). In one embodiment, both R₁ are aralkyl moieties whereinthe alkyl portion of the moiety is substituted with two aryl groups andthe moiety is connected to the molecule via its alkyl group. Forinstance, in one embodiment one or both R₁ is a C₇-C₂₄ aralkyl whereinthe alkyl portion is substituted with two phenyl groups, such as when R₁is 2,2-diphenylethyl or 2,2-dibenzylethyl. In one embodiment, both R₁ offormula (I) is 2,2-diphenylethyl and n is 1, 2 or 5. In one embodiment,each R₁ of formula (I) is 2,2-diphenylethyl, n is 1, 2 or 5 and m and pare each 1.

In one embodiment, at least one R₁ is hydrogen. When one R₁ is hydrogen,the other R₁ may be any moiety listed above for R₁, including an arylgroup such as benzyl. Any of the compounds of formula (I) listed aboveinclude compounds where at least one or both of R₂ is hydrogen or aC₁-C₈ substituted or unsubstituted alkyl. In one embodiment, each R₂ isan unsubstituted alkyl such as methyl. In another embodiment, each R₂ ishydrogen. Any of the compounds of formula (I) listed above may becompounds where q is 1 and m and p are the same. Accordingly, thepolyaminoguanidines of formula (I) may be symmetric with reference tothe polyaminoguanidine core (e.g., excluding R₁). Alternatively, thecompounds of formula (I) may be asymmetric, e.g., when q is 0. In oneembodiment, m and p are 1. In one embodiment, q is 0. In one embodiment,n is an integer from 1 to 5.

In some embodiments, the compound is a polyaminobiguanide or N-alkylatedpolyaminobiguanide. An N-alkylated polyaminobiguanide intends apolyaminobiguanide where at least one imine nitrogen of at least onebiguanide is alkylated. In one embodiment, the compound is apolyaminobiguanide of the formula (I), or a salt, solvate, or hydratethereof, where q is 1, and at least one or each R₁ is of the structure:

where each R₃ is independently selected from the group consisting ofC₁-C₈ alkyl, C₆-C₂₀ aryl, C₆-C₂₀ heteroaryl, C₇-C₂₄ aralkyl, and C₇-C₂₄heteroaralkyl; and each R₂ is independently hydrogen or a C₁-C₈ alkyl.

In one embodiment, in the polyaminobiguanide compound, at least one oreach R₃ is a C₁-C₈ alkyl. For instance, when R₃ is a C₁-C₈ alkyl, thealkyl may be substituted with any substituent, including a primary,secondary, tertiary or quaternary amine. Accordingly, in one embodiment,R₃ is a C₁-C₈ alkyl group substituted with an amine such that R₃ may bee.g., alkyl-NH₂ or an alkyl-amine-alkyl moiety such as—(CH₂)_(y)NH(CH₂)_(z)CH₃ where y and z are independently an integer from1 to 8. In one embodiment, R₃ is —(CH₂)₃NH₂. R₃ may also be a C₄-C₁₅cycloalkyl or a C₃-C₁₅ branched alkyl. In one embodiment, at least oneor each R₃ is a C₆-C₂₀ aryl. In one embodiment, q is I, m and p are 3,and n is 4. In another embodiment, q is I, m and p are 3, and n is 7.

In one embodiment, the compound is a polyaminobiguanide of formula (I)where at least one R₃ is a C₇-C₂₄aralkyl, which in one embodiment is anaralkyl connected to the molecule via its alkyl moiety. In oneembodiment, each R₃ is an aralkyl moiety where the alkyl portion of themoiety is substituted with one or two aryl groups and the moiety isconnected to the molecule via its alkyl moiety. For instance, in oneembodiment at least one or each R₃ is an aralkyl where the alkyl portionis substituted with two phenyl or benzyl groups, such as when R₃ is2,2-diphenylethyl or 2,2-dibenzylethyl. In one embodiment, each R₃ is2,2-diphenylethyl and n is 1, 2 or 5. In one embodiment, each R₃ is2,2-diphenylethyl and n is 1, 2 or 5 and m and p are each 1.

Any of the polyaminobiguanide compounds of formula (I) listed aboveinclude compounds where at least one or both of R₂ is hydrogen or aC₁-C₈ alkyl. In one embodiment, each R₂ is an unsubstituted alkyl, suchas methyl. In another embodiment, each R₂ is a hydrogen.

Any of the polyaminobiguanide compounds of formula (I) listed aboveinclude compounds where q is 1 and m and p are the same. Accordingly,the polyaminobiguanides of formula (I) may be symmetric with referenceto the polyaminobiguanide core. Alternatively, the compounds of formula(I) may be asymmetric. In one embodiment, m and p are 1. In oneembodiment, q is 0. In one embodiment, n is an integer from 1 to 5. Inone embodiment, q, m and p are each 1 and n is 1, 2 or 5.

It is understood and clearly conveyed by this disclosure that each R₁,R₂, R₃, m, n, p and q disclosed in reference to formula (I) intends andincludes all combinations thereof the same as if each and everycombination of R₁, R₂, R₃, m, n, p and q were specifically andindividually listed.

Representative compounds of the formula (I) include, e.g.:

In certain embodiments, the polyamine compound is represented by thestructure according to formula (II):

or a salt, solvate or hydrate thereof,

where n is 1, 2 or 3;

each L is independently a linker of from about 2 to 14 carbons inlength, for example of about 2, 3, 4, 5, 6, 8, 10, 12 or 14 carbon atomsin length, where the linker backbone atoms may be saturated orunsaturated, usually not more than one, two, three, or four unsaturatedatoms will be present in a tether backbone, where each of the backboneatoms may be substituted or unsubstituted (for example with a C₁-C₈alkyl), where the linker backbone may include a cyclic group (forexample, a cyclohex-1,3-diyl group where 3 atoms of the cycle areincluded in the backbone);

each R₁₂ is independently selected from hydrogen and a C₁-C₈ alkyl; and

each R₁₁ is independently selected from hydrogen, C₂-C₈ alkenyl, C₁-C₈alkyl or C₃-C₈ branched alkyl (e.g., methyl, ethyl, tert-butyl,isopropyl, pentyl, cyclobutyl, cyclopropylmethyl, 3-methylbutyl,2-ethylbutyl, 5-NH₂-pent-1-yl, propyl-1-ylmethyl(phenyl)phosphinate,dimethylbicyclo[3.1.1]heptyl)ethyl, 2-(decahydronaphthyl)ethyl and thelike), C₆-C₂₀ aryl or heteroaryl, C₁-C₂₄aralkyl or heteroaralkyl(2-phenylbenzyl, 4-phenylbenzyl, 2-benzylbenzyl, 3-benzylbenzyl,3,3-diphenylpropyl, 3-(benzoimidazolyl)-propyl, 4-isopropylbenzyl,4-fluorobenzyl, 4-tert-butylbenzyl, 3-imidazolyl-propyl, 2-phenylethyland the like), —C(═O)—C₁-C₈ alkyl, —C(═O)—C₁-C₈alkenyl, —C(═O)—C₁-C₈alkynyl, an amino-substituted cycloalkyl (e.g., a cycloalkyl groupsubstituted with a primary, secondary, tertiary or quaternary amine,such as 5-NH₂-cycloheptyl, 3-NH₂-cyclopentyl and the like) and a C₂-C₈alkanoyl (e.g., an alkanoyl substituted with a methyl and an alkylazidegroup).

In certain embodiments, each L is independently selected from:—CHR₁₃—(CH₂)_(m)—, —CHR₁₃—(CH₂)_(n)—CHR₁₃—, —(CH₂)—CHR₁₃—, —CH₂-A-CH₂—and —(CH₂)_(p)—

where:

m is an integer from 1 to 5;

A is (CH₂)_(m), ethane-1,1-diyl or cyclohex-1,3-diyl;

p is an integer from 2 to 14, such as 1, 2, 3, 4 or 5;

n is an integer from 1 to 12; and

R₁₃ is a C₁-C₈alkyl.

A substituted aralkyl or heteroaralkyl with reference to formula (II)intends and includes alkanoyl moieties substituted with an aryl orheteroaryl group, i.e., —C(═O)-aryl, —C(═O)-aralkyl, —C(═O)-heteroaryl,and —C(═O)-heteroaralkyl. In one embodiment, the alkyl portion of thearalkyl or heteroaralkyl moiety is connected to the molecule via itsalkyl moiety. For instance at least one or both of R₁₁ may be an aralkylmoiety such as 2-phenylbenzyl, 4-phenylbenzyl, 3,3,-diphenylpropyl,2-(2-phenylethyl)benzyl, 2-methyl-3-phenylbenzyl, 2-napthylethyl,4-(pyrenyl)butyl, 2-(3-methylnapthyl)ethyl,2-(1,2-dihydroacenaphth-4-yl)ethyl and the like. In another embodiment,at least one or both of R₁₁ may be a heteroaralkyl moiety such as3-(benzoimidazolyl)propanoyl, 1-(benzoimidazolyl)methanoyl,2-(benzoimidazolyl)ethanoyl, 2-(benzoimidazolyl)ethyl and the like.

In certain embodiments, the compound of formula (II) comprises at leastone moiety selected from the group consisting of t-butyl, isopropyl,2-ethylbutyl, 1-methylpropyl, 1-methylbutyl, 3-butenyl, isopent-2-enyl,2-methylpropan-3-olyl, ethylthiyl, phenylthiyl, propynoyl,1-methyl-1H-pyrrole-2-yl; trifluoromethyl, cyclopropanecarbaldehyde,halo-substituted phenyl, nitro-substituted phenyl, alkyl-substitutedphenyl, 2,4,6-trimethylbenzyl, halo-5-substituted phenyl (such aspara-(F₃S)-phenyl, azido and 2-methylbutyl.

In certain embodiments, in formula (II), each R₁₁ is independentlyselected from hydrogen, n-butyl, ethyl, cyclohexylmethyl,cyclopentylmethyl, cyclopropylmethyl, cycloheptylmethyl,cyclohexyleth-2-yl, and benzyl.

In certain embodiments, the polyamine compound is of the structure offormula (II), where n is 3, such that the compound has a structureaccording to formula (III):

where L₁, L₂ and L₃ are independently selected from —CHR₁₃—(CH₂)_(m)—,—CHR₁₃—(CH₂)_(n)—CHR₁₃—, —(CH₂)_(m)—CHR₁₃—, —CH₂-A-CH₂— and —(CH₂)_(p)—

where m, A, p, n and R₁₃ are as defined above.

In certain embodiments, the polyamine compound is of the structure ofFormula (III) where: L₁ is —CHR₁₃—(CH₂)_(m)—; L₂ is—CHR₁₃—(CH₂)_(n)—CHR₁₃—; and L₃ is —(CH₂)_(m)—CHR₁₃—; where R₁₁, R₁₂,R₁₃, m and n are as defined above.

In certain embodiments, the polyamine compound is of the structure ofFormula (III) where: L₁, L₂ and L₃ are independently —CH₂-A-CH₂—; andR₁₂ is hydrogen; where R₁₁ and A are as defined above. In particularembodiments, at least one of an A and an R₁₁ comprises an alkenylmoiety.

In certain embodiments, the polyamine compound is of the structure ofFormula (III) where: L₁, L₂ and L₃ are independently —(CH₂)_(p)— where pis as defined above; and R₁₂ is hydrogen. In particular embodiments, forL₁ and L₃, p is an integer from 3 to 7, and for L₃ p is an integer from3 to 14.

In certain embodiments, the polyamine compound is of the structure ofFormula (III) where: L₁, and L₃ are independently —(CH₂)_(p)—; L₂ is—CH₂-A-CH₂—; and R₁₂ is hydrogen; where R₁₂, p and A are as definedabove. In particular embodiments, for L₁ and L₃, p is an integer from 2to 6, and for L₃ A is (CH₂)^(x) where x is an integer from 1 to 5, orcyclohex-1,3-diyl.

In certain embodiments, the polyamine compound is of the structure ofFormula (II), where n is 2, such that the compound has a structureaccording to formula (IV):

where L₁ and L₂ are independently selected from—CHR₁₃—(CH₂)_(m)—CHR₁₃—(CH₂)_(n)—CHR₁₃—, —(CH₂)_(n), CHR₁₃—, —CH₂-A-CH₂—and —(CH₂)_(p)—

where m, A, p, n, and R₁₃ are as defined above.

In certain embodiments, the polyamine compound is of the structure ofFormula (IV) where: L₁ is —(CH₂)_(p)—; and L₂ is —(CH₂)_(m)—CHR₁₃—;where R₁₃, m and p are as defined above. In particular embodiments, forL₁ p is an integer from 3 to 10, and for L₂ n is an integer from 2 to 9.

In certain embodiments, the polyamine compound is of the structure ofFormula (IV) where: L₁ and L₂ are —(CH₂)_(p)—; where p is as definedabove. In particular embodiments, p is an integer from 3 to 7.

In certain embodiments, the polyamine compound is of the structure ofFormula (II), where n is 1, such that the compound has a structureaccording to formula (V):

where L₁ is —(CH₂)_(p)— where p is as defined above. In particularembodiments, p is an integer from 2 to 6.

In particular embodiments, in formula (V), one R₁₁ is anamino-substituted cycloalkyl (e.g., a cycloalkyl group substituted witha primary, secondary, tertiary or quaternary amine) or a C₂-C₈ alkanoyl(which alkanoyl may be substituted with one or more substituents such asa methyl or an alkylazide group); and the other R₁₁ is a C₁-C₈ alkyl ora C₇-C₂₄ aralkyl.

Representative compounds of the formula (II) include, e.g.:

Phenylcyclopropylamine derivatives that are inhibitors of includecompounds represented by formula (VI):

wherein:

each of R1-R5 is independently selected from H, halo, alkyl, alkoxy,cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl,-L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl,amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl,arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl,hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate,nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato,trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, and C-amido;

R6 is H or alkyl;

R7 is H, alkyl, or cycloalkyl;

R8 is an -L-heterocyclyl wherein the ring or ring system of the-L-heterocyclyl has from 0 to 3 substituents selected from halo, alkyl,alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl,-L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl,amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl,arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl,hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate,nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato,trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, and C-amido; or

R8 is -L-aryl wherein the ring or ring system of the -L-aryl has from 1to 3 substituents selected from halo, alkyl, alkoxy, cycloalkoxy,haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl,acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino,alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy,aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl,heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate, nitro,sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato,trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, and C-amido;

where each L is independently selected from —(CH₂)_(n)—(CH₂)_(n)—,—(CH₂)_(n)NH(CH₂)_(n)—, —(CH₂)_(n)O(CH₂)_(n)—, and—(CH₂)_(n)S(CH₂)_(n)—, and where each n is independently chosen from 0,1, 2, and 3;

or a pharmaceutically acceptable salt thereof.

In some cases, L is a covalent bond. In some cases, R6 and R7 are hydro.In some cases, one of R1-R5 is selected from -L-aryl, -L-heterocyclyl,and -L-carbocyclyl.

In some embodiments of the compound of Formula VI, the substituent orsubstituents on the R8 ring or ring system is/are selected fromhydroxyl, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy,—N(C₁-3 alkyl)₂, —NH(C₁-3 alkyl), —C(═O)NH₂, —C(═O)NH(C₁-3 alkyl),—C(═O)N(C₁-3 alkyl)₂, —S(═O)₂(C₁-3 alkyl), —S(═O)₂NH₂, —S(O)₂NH₂,—S(O)₂N(C₁-3 alkyl)₂, —S(═O)₂NH(C₁-3 alkyl), —CN, —NH₂, and —NO₂.

In certain embodiments, a compound of the invention is of formula (VI)where:

each R1-R5 is optionally substituted and independently chosen from —H,halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl,-L-heteroaryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy,alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyl, arylalkenyl,arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano,cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy,isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide,thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, and C-amido;

R6 is chosen from —H and alkyl;

R7 is chosen from —H, alkyl, and cycloalkyl;

R8 is chosen from —C(═O)NRxRy and —C(═O)Rz;

Rx when present is chosen from —H, alkyl, alkynyl, alkenyl,-L-carbocyclyl, -L-aryl, and -L-heterocyclyl, all of which areoptionally substituted (except —H);

Ry when present is chosen from —H, alkyl, alkynyl, alkenyl,-L-carbocyclyl, -L-aryl, and -L-heterocyclyl, all of which areoptionally substituted (except —H), where Rx and Ry may be cyclicallylinked;

Rz when present is chosen from —H, alkoxy, -L-carbocyclyl,-L-heterocyclyl, -L-aryl, wherein the aryl, heterocyclyl, or carbocyclylare optionally substituted; each L is a linker that links the mainscaffold of Formula I to a carbocyclyl, heterocyclyl, or aryl group,wherein the hydrocarbon portion of the linker -L- is saturated,partially saturated, or unsaturated, and is independently chosen from asaturated parent group having a formula of —(CH₂)_(n)—(CH₂)_(n)—,—(CH₂)_(n)C(═O)(CH₂)—, —(CH₂)_(n)C(═O)NH(CH₂)_(n)—,—(CH₂)_(n)NHC(O)O(CH₂)_(n)—, —(CH₂)_(n)NHC(═O)NH(CH₂)_(n)—,—(CH₂)_(n)NHC(═S)S(CH₂)_(n)—, —(CH₂)_(n)OC(═O)S(CH₂)_(n)—,—(CH₂)_(n)NH(CH₂)_(n)—, —(CH₂)_(n)—O—(CH₂)_(n)—, —(CH₂)_(n)S(CH₂)_(n)—,and —(CH₂)_(n)NHC(═S)NH(CH₂)_(n)—, where each n is independently chosenfrom 0, 1, 2, 3, 4, 5, 6, 7, and 8. According to this embodiment,optionally substituted refers to zero or 1 to 4 optional substituentsindependently chosen from acylamino, acyloxy, alkenyl, alkoxy,cycloalkoxy, alkyl, alkylthio, cycloalkylthio, alkynyl, amino, aryl,arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio,heteroarylthio, carbocyclyl, cyano, cyanato, halo, haloalkyl, haloaryl,hydroxyl, heteroaryl, heteroaryloxy, heterocyclyl, heteroarylalkoxy,isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide,thiocarbonyl, thiocyanato, trihalomethanesulfonamido, 0-carbamyl,N-carbamyl, 0-thiocarbamyl, N-thiocarbamyl, and C-amido. In a morespecific aspect of this embodiment, the optional substituents are 1 or 2optional substituents chosen from halo, alkyl, aryl, and arylalkyl.

In certain embodiments, in formula (VI), R8 is —CORz, such that thecompound is of the following structure:

where: R1-R7 are described above; and Rz is -L-heterocyclyl which isoptionally substituted with from 1-4 optional substituents independentlychosen from acylamino, acyloxy, alkenyl, alkoxy, cycloalkoxy, alkyl,alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyl, arylalkenyl,arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, carbocyclyl,cyano, cyanato, halo, haloalkyl, haloaryl, hydroxyl, heteroaryl,heteroaryloxy, heterocyclyl, heteroarylalkoxy, isocyanato,isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl,thiocyanato, trihalomethanesulfonamido, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, and C-amido, and wherein said -L- isindependently chosen from —(CH₂)_(n)—(CH₂)_(n)—, —(CH₂)_(n)NH(CH₂)_(n)—,—(CH₂)_(n)—O—(CH₂)_(n)—, and —(CH₂)_(n)S(CH₂)_(n)—, where each n isindependently chosen from 0, 1, 2, and 3.

In a specific aspect of this embodiment, each L is independently chosenfrom —(CH₂)_(n)—(CH₂)_(n)— and —(CH₂)_(n)—O—(CH₂)_(n) where each n isindependently chosen from 0, 1, 2, and 3. In a more specific aspect ofthis embodiment, each L is chosen from a bond, —CH₂—, —CH₂CH₂—, —OCH₂—,—OCH₂CH₂—, —CH₂OCH₂—, —CH₂CH₂CH₂—, —OCH₂CH₂CH₂—, and —CH₂OCH₂CH₂—. In aneven more specific aspect, each L is chosen from a bond, —CH₂—,—CH₂CH₂—, OCH₂—, and —CH₂CH₂CH₂—. In yet an even more specific aspect, Lis chosen from a bond and —CH₂—.

Exemplary compounds of Formula VI include:

Exemplary compounds of Formula VI include:N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;2-{[(trans)-2-phenylcyclopropyl]amino acetamide;N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}propanamide;2-{[(trans)-2-phenylcyclopropyl]amino}-N-prop-2-ynylacetamide;N-isopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;N-(tert-butyl)-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;N-(2-morpholin-4-yl-2-oxoethyl)-N-[(trans)-2-phenylcyclopropyl]amine;2-{[(trans)-2-phenylcyclopropyl]amino}propanamide; methyl2-{[(trans)-2-phenylcyclopropyl]amino}propanoate;N-cyclopropyl-2-{methyl[(trans)-2-phenylcyclopropyl]amino}acetamide;2-{methyl[(trans)-2-phenylcyclopropyl]amino}acetamide;N-methyl-trans-2-(phenylcyclopropylamino)propanamide;1-(4-methylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;1-(4-ethylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;1-(4-benzylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)-ethanone;2-((trans)-2-phenylcyclopropylamino)-1-(4-phenylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)-N-cyclopropylacetamide;2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)-1-(4--methylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(3-chlorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;2-((trans)-2-(biphenyl-4-yl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;1-(4-methylpiperazin-1-yl)-2-((trans)-2-(4-phenethoxyphenyl)cyclopropylamino)ethanone;2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(biphenyl-4-ylmethoxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;(trans)-N-(4-fluorobenzyl)-2-phenylcyclopropanamine;(trans)-N-(4-fluorobenzyl)-2-phenylcyclopropanaminiurn;4-(((trans)-2-phenylcyclopropylamino)methyl)benzonitrile;(trans)-N-(4-cyanobenzyl)-2-phenylcyclopropanaminium;(trans)-2-phenyl-N-(4-(trifluoromethyl)benzyl)cyclopropanamine;(trans)-2-phenyl-N-(4-(trifluoromethyl)benzyl)cyclopropanaminium;(trans)-2-phenyl-N-(pyridin-2-ylmethyl)cyclopropanamine;(trans)-2-phenyl-N-(pyridin-3-ylmethyl)cyclopropanamine;(trans)-2-phenyl-N-(pyridin-4-ylmethyl)cyclopropanamine;(trans)-N-((6-methylpyridin-2-yl)methyl)-2-phenylcyclopropanamine;(trans)-2-phenyl-N-(thiazol-2-ylmethyl)cyclopropanamine;(trans)-2-phenyl-N-(thiophen-2-ylmethyl)cyclopropanamine;(trans)-N-((3-bromothiophen-2-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((4-bromothiophen-2-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-(3,4-dichlorobenzyl)-2-phenylcyclopropanamine;(trans)-N-(3-fluorobenzyl)-2-phenylcyclopropanaminium;(trans)-N-(2-fluorobenzyl)-2-phenylcyclopropanamine;(trans)-2-phenyl-N-(quinolin-4-ylmethyl)cyclopropanaraine;(trans)-N-(3-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-2-phenyl-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)cyclopropanamine;(trans)-N-((6-chloropyridin-3-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((4-methylpyridin-2-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((6-methoxypyridin-2-yl)methyl)-2-phenylcyclopropanamine;2-(((trans)-2-phenylcyclopropylamino)methyl)pyridin-3-ol;(trans)-N-((6-bromopyridin-2-yl)methyl)-2-phenylcyclopropanamine;4-(((trans)-2-(4(benzyloxy)phenyl)cyclopropylamino)methyl)benzonitrile;(trans)-N-(4-(benzyloxy)benzyl)-2-phenylcyclopropanamine;(trans)-N-benzyl-2-(4-(benzyloxy)phenyl)cyclopropanamine;(trans)-2-(4-(benzyloxy)phenyl)-N-(4-methoxybenzyl)cyclopropanamine;(trans)-2-(4-(benzyloxy)phenyl)-N-(4-fluorobenzyl)cyclopropanamine-;(trans)-2-phenyl-N-(quinolin-2-ylmethyl)cyclopropanamine;(trans)-2-phenyl-N-((5-(trifluoromethyl)pyridin-2-yl)methyl)cyclopropanamine;(trans)-N-((3-fluoropyridin-2-yl)methyl)-2-phenylcyclopropanamine;(trans)-2-phenyl-N-(quinolin-3-ylmethyl)cyclopropanamine;(trans)-N-((6-methoxypyridin-3-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((5-methoxypyridin-3-yl)methyl)-2-phenylcyclopropanamine-;(trans)-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((3H-indol-3-yl)methyl)-2-phenylcyclopropanamine;3-(((trans)-2-phenylcyclopropylamino)methyl)benzonitrile;(trans)-N-(2-methoxybenzyl)-2-phenylcyclopropanamine;3-(((trans)-2-phenylcyclopropylamino)methyl)pyridin-2-amine;(trans)-N-((2-chloropyridin-3-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-(3,4-dimethoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-phenylcyclopropanamine;(trans)-N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-2-phenyl-cyclopropanamine;(trans)-N-(2,6-difluoro-4-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-2-phenyl-N-(4-(trifluoromethoxy)benzyl)cyclopropanamine;(trans)-N-(5-fluoro-2-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(2-fluoro-4-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-((4-methoxynaphthalen-1-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-(2-fluoro-6-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-((2-methoxynaphthalen-1-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((4,7-dimethoxynaphthalen-1-yl)methyl)-2-phenylcyclopropanamine-;(trans)-N-(4-methoxy-3-methylbenzyl)-2-phenylcyclopropanamine;(trans)-N-(3-chloro-4-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(3-fluoro-4-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(4-methoxy-2-methylbenzyl)-2-phenylcyclopropanamine;(trans)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-6-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-((2,2-dimethylchroman-6-yl)methyl)-2-phenylcyclopropanamine;(trans)-N-(4-methoxy-2,3-dimethylbenzyl)-2-phenylcyclopropanamine;(trans)-N-(4-methoxy-2,5-dimethylbenzyl)-2-phenylcyclopropanamine;(trans)-N-(2-fluoro-4,5-dimethoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(3-chloro-4,5-dimethoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(2-chloro-3,4-dimethoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(2,4-dimethoxy-6-methylbenzyl)-2-phenylcyclopropanamine;(trans)-N-(2,5-dimethoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(2,3-dimethoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-(2-chloro-3-methoxybenzyl)-2-phenylcyclopropanamine;(trans)-N-((1H-indol-5-yl)methyl)-2-phenylcyclopropanamine;(trans)-2-(4-(benzyloxy)phenyl)-N-(pyridin-2-ylmethyl)cyclopropanamine;(trans)-2-(4-(benzyloxy)phenyl)-N-(2-methoxybenzyl)cyclopropanamine;(trans)-N-(1-(4-methoxyphenyl)ethyl)-2-phenylcyclopropanaraine;(trans)-N-(1-(3,4-dimethoxyphenyl)ethyl)-2-phenylcyclopropanamine;(trans)-N-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethyl)-2-phenylcyclopropanamine;(trans)-N-(1-(5-fluoro-2-methoxyphenyl)ethyl)-2-phenylcyclopropanamine;(trans)-N-(1-(3,4-dimethoxyphenyl)propan-2-yl)-2-phenylcyclopropanamine;(trans)-N-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)-2-phenylcyclopropanamine;

and pharmaceutically acceptable salts thereof.

Alternative small molecule LSD inhibitor compounds may be selected fromselective LSD1 and LSD1/MAOB dual inhibitors disclosed for example inWO2010/043721 (PCT/EP2009/063685), WO2010/084160 (PCT/EP2010/050697),PCT/EP2010/055131; PCT/EP2010/055103; and EP application numberEP10171345 all of which are explicitly incorporated herein by referencein their entireties to the extent they are not inconsistent with theinstant disclosure. Representative compounds of this type includephenylcyclopropylamine derivatives or homologs, illustrative examples ofwhich include phenylcyclopropylamine with one or two substitutions onthe amine group; phenylcyclopropylamine with zero, one or twosubstitutions on the amine group and one, two, three, four, or fivesubstitution on the phenyl group; phenylcyclopropylamine with one, two,three, four, or five substitution on the phenyl group;phenylcyclopropylamine with zero, one or two substitutions on the aminegroup wherein the phenyl group of PCPA is substituted with (exchangedfor) another ring system chosen from aryl or heterocyclyl to give anaryl- or heteroaryl-cyclopropylamine having zero, one or twosubstituents on the amine group; phenylcyclopropylamine wherein thephenyl group of PCPA is substituted with (exchanged for) another ringsystem chosen from aryl or heterocyclyl to give an aryl- orheterocyclyl-cyclopropylamine wherein said aryl- orheterocyclyl-cyclopropylamine on said aryl or heterocyclyl moiety haszero, one or two substitutions on the amine group and one, two, three,four, or five substitution on the phenyl group; phenylcyclopropylaminewith one, two, three, four, or five substitution on the phenyl group; orany of the above described phenylcyclopropylamine analogs or derivativeswherein the cyclopropyl has one, two, three or four additionalsubstituents. Suitably, the heterocyclyl group described above in thisparagraph in a heteroaryl.

Non-limiting embodiments of phenylcyclopropylamine derivatives oranalogs include “cyclopropylamine amide” derivatives and“cyclopropylamine” derivatives. Specific examples of “cyclopropylamineacetamide” derivatives include, but are not limited to:N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}propanamide;2-{[(trans)-2-phenylcyclopropyl]amino}-N-prop-2-ynylacetamide;N-isopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;N-(tert-butyl)-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;N-(2-morpholin-4-yl-2-oxoethyl)-N-[(trans)-2-phenylcyclopropyl]amine;2-{[(trans)-2-phenylcyclopropyl]amino}propanamide; Methyl2-{[(trans)-2-phenylcyclopropyl]amino}propanoate;1-(4-methylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;1-(4-ethylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;1-(4-benzylpiperazin-1-yl)-2-((trans)-2-phenylcyclopropylamino)ethanone;2-((trans)-2-phenylcyclopropylamino)-1-(4-phenylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;2-((trans)-2-(1,1′-biphenyl-4-yl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)-N-cyclopropylacetamide;2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)-1-(4--methylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;2-((trans)-2-(4-(3-chlorobenzyloxy)phenyl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;1-(4-methylpiperazin-1-yl)-2-((trans)-2-(4-phenethoxyphenyl)cyclopropylamino)ethanone;2-((trans)-2-(biphenyl-4-yl)cyclopropylamino)-1-(4-methylpiperazin-1-yl)ethanone;N-cyclopropyl-2-{[(trans)-2-phenylcyclopropyl]amino}acetamide;N-methyl-trans-2-(Phenylcyclopropylamino)propanamide;2-{methyl[(trans)-2-phenylcyclopropyl]amino}acetamide;N-[2-(4-methylpiperazin-1-yl)ethyl]-N-[(trans)-2-phenylcyclopropyl]amine;N-cyclopropyl-N′-[(trans)-2-phenylcyclopropyl]ethane-1,2-diamine;N,N-dimethyl-N′-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)ethane-1,2-diamine;(3R)-1-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)pyrrolidin-3-amine;(3S)—N,N-dimethyl-1-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)pyrrolidin-3-amine;(3R)—N,N-dimethyl-1-(2-{[(trans)-2-phenylcyclopropyl]amino}ethyl)pyrrolidin-3-amine;N-[(trans)-2-phenylcyclopropyl]-N-(2-piperazin-1-ylethyl)amine;N,N-diethyl-N′-[(trans)-2-phenylcyclopropyl]ethane-1,2-diamine;N-[(trans)-2-phenylcyclopropyl]-N-(2-piperidin-1-ylethyl)amine;(trans)-2-(4-(benzyloxy)phenyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)cyclopropanamine;(trans)-N-(2-(4-methylpiperazin-1-yl)ethyl)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine;(trans)-2-(3′-chlorobiphenyl-4-yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)cyclopropanamine;(R)-1-(2-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropylamino)ethyl)pyrrolidin-3-amine;andN¹-cyclopropyl-N²-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl-)cyclopropyl)ethane-1,2-diamine.

Specific examples of “cyclopropylamine” derivatives, include, but arenot limited to:N-4-fluorobenzyl-N-{(trans)-2-[4-(benzyloxy)phenyl]cyclopropyl}amine,N-4-methoxybenzyl-N-{(trans)-2-[4-(benzyloxy)phenyl]cyclopropyl}amine,N-benzyl-N-{(trans)-2-[4-(benzyloxy)phenyl]cyclopropyl}amine,N-[(trans)-2-phenylcyclopropyl]amino-methyl)pyridin-3-ol,N-[(trans)-2-phenylcyclopropyl]-N-(3-methylpyridin-2-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(4-chloropyridin-3-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(4-trifluoromethylpyridin-3-yl-methyl)amine,N-(3-methoxybenzyl)-N-[(trans)-2-phenylcyclopropyl]amine,N-[(trans)-2-phenylcyclopropyl]-N-(quinolin-4-ylmethyl)amine,N-(2-fluorobenzyl)-N-[(trans)-2-phenylcyclopropyl]amine,N-(3-fluorobenzyl)-N-[(trans)-2-phenylcyclopropyl]amine,N-[(trans)-2-phenylcyclopropyl]-N-(3,4-dichloro-1-phenylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(5-bromo-thiophen-2-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(3-bromo-thiophen-2-ylmethyl)-amine,N-[(trans)-2-phenylcyclopropyl]-N-(thiophen-2-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(1,3-thiazol-2-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(3-methyl-pyridin-2-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(pyridin-4-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(pyridin-3-ylmethyl)amine,N-[(trans)-2-phenylcyclopropyl]-N-(pyridin-2-ylmethyl)amine,[(trans)-2-phenylcyclopropyl]-N-[4-(trifluoromethyl)benzyl]amine,({[(trans)-2-phenylcyclopropyl]amino}methyl)benzonitrile,N-(4-fluorobenzyl)-N-[(trans)-2-phenylcyclopropyl]amine,N-[(trans)-2-phenylcyclopropyl]-N-(3-bromo-pyridin-2-ylmethyl)amine,N-4-cyanobenzyl-N-{(trans)-2-[4-(benzyloxy)phenyl]cyclopropyl}amine,N-4-[(benzyloxy)-benzyl]-N-[(trans)-2-(4-phenyl)cyclopropyl]amine;2-((trans)-2-(4-(4-cyanobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(3-cyanobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(4-fluorobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(3-fluorobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(3-chlorobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(4-chlorobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(3-bromobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-(3,5-difluorobenzyloxy)phenyl)cyclopropylamino)acetamide,2-((trans)-2-(4-phenethoxyphenyl)cyclopropylamino)acetamide,2-((trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropylamino)acetamide,and 2-((trans)-2-(3′-chlorobiphenyl-4-yl)cyclopropylamino)acetamide.

Other examples of LSD1 inhibitors are, e.g., phenelzine or pargyline(propargylamine) or a derivative or analog thereof. Derivatives andanalogs of phenelzine and pargyline (propargylamine) include, but arenot limited to, compounds where the phenyl group of the parent compoundis replaced with a heteroaryl or optionally substituted cyclic group orthe phenyl group of the parent compound is optionally substituted with acyclic group. In one aspect, the phenelzine or pargyline derivative oranalog thereof has selective LSD1 or dual LSD1/MAOB inhibitory activityas described herein. In some embodiments, the phenelzine derivative oranalog has one, two, three, four or five substituents on the phenylgroup. In one aspect, the phenelzine derivative or analog has the phenylgroup substituted with (exchanged for) an aryl or heterocyclyl groupwherein said aryl or heterocyclyl group has zero, one, two, three, fouror five substituents. In one aspect, the pargyline derivative or analoghas one, two, three, four or five substituents on the phenyl group. Inone aspect, the pargyline derivative or analog has the phenyl groupsubstituted with (exchanged for) an aryl or heterocyclyl group whereinsaid aryl or heterocyclyl group has zero, one, two, three, four or fivesubstituents. Methods of preparing such compounds are known to theskilled artisan.

The present invention also contemplates tranylcypromine derivatives asdescribed for example by Binda et al. (2010. J. Am. Chem. Soc.132:6827-6833, which is hereby incorporated by reference herein in itsentirety) as inhibitors of LSD (e.g., LSD1 and/or LSD2) enzymaticfunction. Non-limiting example of such compounds include:

Alternatively, LSD1 inhibitor compounds may be selected fromtranylcypromine analogs described by Benelkebir et al. (2011. Bioorg.Med. Chem. doi:10.1016/j.bmc.2011.02.017, which is hereby incorporatedby reference herein in its entirety), Representative analogs of thistype, including o-, m- and p-bromo analogues include:(1R,2S)-2-(4-bromophenyl)cyclopropanamine hydrochloride (Compound 4c),(1R,2S)-2-(3-bromophenyl)cyclopropanamine hydrochloride (Compound 4d),(1R,2S)-2-(2-bromophenyl)cyclopropanamine hydrochloride (Compound 4e),(1R,2S)-2-(biphenyl-4-yl)cyclopropanamine hydrochloride (Compound 4f).

Reference also may be made to peptide scaffold compounds disclosed byCulhane et al. (2010. J. Am. Chem. Soc. 132:3164-3176, which is herebyincorporated by reference herein in its entirety), which includechlorovinyl, endo-cyclopropylamine, and hydrazine functionalities.Non-limiting compounds disclosed by Culhane et al. includepropargyl-Lys-4, N-methylpropargyl-Lys-4 H3-21, cis-3-chloroallyl-Lys-4H3-21, trans-3-chloroallyl-Lys-4 H3-21, exo-cyclopropyl-Lys-4 H3-21,endo-cyclopropyl-Lys-4 H3-21, endo-dimethylcyclopropyl-Lys-4,hydrazino-Lys-4 H3-21 and hydrazino-Lys-4 H3-21.

Alternative cyclopropylamine compounds that are useful for inhibitingLSD1 include those disclosed by Fyfe et al. in U.S. Publication No.2013/0197013, which is incorporated herein by reference in its entirety.Illustrative cyclopropylamine inhibitors of LSD1, which are disclosed asbeing selective for inhibiting LSD1, include compounds according toformula VII:

wherein:

E is —N(R3)-, —O—, or —S—, or is —X³═X⁴—;

X¹ and X² are independently C(R2) or N;

X³ and X⁴, when present, are independently C(R2) or N;

(G) is a cyclyl group (as shown in formula VII, the cyclyl group (G) hasn substituents (R1));

each (R1) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl;

each (R2) is independently chosen from —H, alkyl, alkenyl, alkynyl,cyclyl, -L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has1, 2, or 3 independently chosen optional substituents or two (R2) groupscan be taken together to form a heterocyclyl or aryl group having 1, 2,or 3 independently chosen optional substituents, wherein said optionalsubstituents are independently chosen from alkyl, alkanoyl, heteroalkyl,heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,heterocyclylalkoxy, aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy,oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl,amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;

R3 is —H or a (C₁-C₆)alkyl group;

each L1 is independently alkylene or heteroalkylene; and

-   -   n is 0, 1, 2, 3, 4 or 5,

or an enantiomer, a diastereomer, or a mixture thereof, or apharmaceutically acceptable salt or solvate thereof.

In some embodiments, compounds of formula VII are represented by formulaVIII:

wherein:

X¹ is CH or N; (G) is a cyclyl group;

each (R1) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl;

each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has1, 2, or 3 optional substituents, wherein said optional substituents areindependently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl,haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy, heterocyclylalkoxy,aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy,carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino,aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;

each L1 is independently alkylene or heteroalkylene;

-   -   m is 0, 1, 2 or 3; and n is 0, 1, 2, 3, 4 or 5, provided that n        and m are chosen independently such that n+m is greater than        zero when X¹ is —CH— and (G) is an aryl,

or an enantiomer, a diastereomer, or a mixture thereof, or apharmaceutically acceptable salt or solvate thereof.

In other embodiments, compounds of formula VII are represented byformula IX:

wherein:

(G) is a cyclyl group;

each (R1) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl;

each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has0, 1, 2, or 3 optional substituents, wherein said optional substituentsare independently chosen from alkyl, alkanoyl, heteroalkyl,heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,heterocyclylalkoxy, aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy,oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl,amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;

each L1 is independently alkylene or heteroalkylene; m is 0, 1, 2 or 3;and

-   -   n is 0, 1, 2, 3, 4 or 5,

or an enantiomer, a diastereomer, or a mixture thereof, or apharmaceutically acceptable salt or solvate thereof.

In still other embodiments, compounds of formula VII are represented byformula X:

wherein:

E is —N(R3)-, —O—, or —S—, or is —X³═X⁴—;

X¹, X², X³ and X⁴ are independently C(R2) or N, provided that at leastone of X¹, X², X³ and X⁴ is N when E is —X³═X⁴—;

(G) is a cyclyl group; each (R1) is independently chosen from alkyl,alkenyl, alkynyl, cyclyl, -L1-cyclyl, -L1-amino, -L1-hydroxyl, amino,amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl,sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;

each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has1, 2, or 3 optional substituents, wherein said optional substituents areindependently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl,haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy, heterocyclylalkoxy,aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy,carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino,aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;

R3 is —H or a (C₁-C₆)alkyl group; each L1 is alkylene or heteroalkylene;and n is 0, 1, 2, 3, 4 or 5,

or an enantiomer, a diastereomer, or a mixture thereof, or apharmaceutically acceptable salt or solvate thereof.

In still other embodiments, compounds of formula VII are represented byformula XI:

wherein:

X¹, X², X³ and X⁴ are independently CH or N, provided that at least oneof X¹, X², X³ and X⁴ is N;

(G) is a cyclyl group; each (R1) is independently chosen from alkyl,alkenyl, alkynyl, cyclyl, -L1-cyclyl, -L1-amino, -L1-hydroxyl, amino,amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl,sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;

each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl,-L1-cyclyl, -L1-amino, -L1-hydroxyl, amino, amido, nitro, halo,haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl,alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has1, 2, or 3 optional substituents, wherein said optional substituents areindependently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl,haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy, heterocyclylalkoxy,aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy,carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino,aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio,sulfonamide, sulfinyl, sulfonyl, urea, or carbamate; each L1 is alkyleneor heteroalkylene;

-   -   m is 0, 1, 2 or 3; and n is 0, 1, 2, 3, 4 or 5,

or an enantiomer, a diastereomer, or a mixture thereof, or apharmaceutically acceptable salt or solvate thereof.

Representative compounds according to formula VII are suitably selectedfrom: (trans)-2-(3′-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine;(trans)-2-(terphenyl-4-yl)cyclopropanamine;4′-((trans)-2-aminocyclopropyl)biphenyl-4-ol;4′-((trans)-2-aminocyclopropyl)biphenyl-3-ol;(trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(Trans)-2-(6-(3,5-dichlorophenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(4-chlorophenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(3-chlorophenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(4-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(4-methoxyphenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(3-methoxyphenyl)pyridin-3-yl)cyclopropanamine;4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzonitrile;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzonitrile;(Trans)-2-(6-p-tolylpyridin-3-yl)cyclopropanamine;(Trans)-2-(6-m-tolylpyridin-3-yl)cyclopropanamine;4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzamide;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzamide;2-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenol;(Trans)-2-(6-(3-methoxy-4-methylphenyl)pyridin-3-yl)cyclopropanamine;5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-fluorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-fluorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-fluorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-fluorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2,4-difluorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2,4,6-trifluorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-chlorophenol;(Trans)-2-(6-(2-fluoro-3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(Trans)-2-(6-(5-chlorothiophen-2-yl)pyridin-3-yl)cyclopropanamine;(Trans)-2-(6-(5-methylthiophen-2-yl)pyridin-3-yl)cyclopropanamine;(Trans)-2-(6-(1H-indol-6-yl)pyridin-3-yl)cyclopropanamine;(Trans)-2-(6-(benzo[b]thiophen-5-yl)pyridin-3-yl)cyclopropanamine;3-(5-((trans)-2-aminocyclopropyl)-3-methylpyridin-2-yl)phenol;(trans)-2-(6-(3-chlorophenyl)-5-methylpyridin-3-yl)cyclopropanamine;(trans)-2-(5-methyl-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(4-fluoro-3-methoxyphenyl)pyridin-3-yl)cyclopropanamine,(trans)-2-(6-(3-fluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(2-fluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine,(trans)-2-(6-(2-fluoro-3-methoxyphenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(3-chloro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(2-chloro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(3-methoxy-5-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-methoxybenzonitrile;5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-methylphenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-chlorophenol;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenol;(trans)-2-(6-(2-fluoro-5-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(2-chloro-5-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(3,5-bis(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)acetamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)methanesulfonamide;(trans)-2-(6-(benzo[b]thiophen-2-yl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(benzo[b]thiophen-3-yl)pyridin-3-yl)cyclopropanamine;5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)thiophene-2-carbonitrile;(trans)-2-(6-(4-methylthiophen-3-yl)pyridin-3-yl)cyclopropanamine;(trans)-2-(2-chloro-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;(trans)-2-(2-(4-chlorophenyl)-6-(3-(trifluoromethyl)phenyl)pyridine-3-yl)cyclopropanamine;4-(3-((trans)-2-aminocyclopropyl)-6-(3-(trifluoromethyl)phenyl)pyridin-2-yl)phenol;4-(3-((trans)-2-aminocyclopropyl)-6-(3-(trifluoromethyl)phenyl)-pyridin-2-yl)benzamide;(trans)-2-(2-methyl-6-(3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropanamine;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-hydroxybenzonitrile;(trans)-2-(6-(3,4-difluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2,3-difluorophenol;(trans)-2-(6-(3-chloro-4-fluoro-5-methoxyphenyl)pyridin-3-yl)cyclopropanamine;5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-3-chloro-2-fluorophenol;(trans)-2-(6-(1H-indazol-6-yl)pyridin-3-yl)cyclopropanamine;(trans)-2-(6-(9H-carbazol-2-yl)pyridin-3-yl)cyclopropanamine;6-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)indolin-2-one;6-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)benzofuran-2(3H)-one;4-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)pyridin-2(1H)-one;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)benzenesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)propane-2-sulfonamide;4′-((trans)-2-aminocyclopropyl)-4-fluorobiphenyl-3-ol;4′-((trans)-2-aminocyclopropyl)-5-chlorobiphenyl-3-ol;4′-((trans)-2-aminocyclopropyl)-5-chloro-4-fluorobiphenyl-3-ol;N-(4′-((trans)-2-aminocyclopropyl)biphenyl-3-yl)benzenesulfonamide;N-(4′-((trans)-2-aminocyclopropyl)biphenyl-3-yl)propane-2-sulfonamide;N-(4′-((trans)-2-aminocyclopropyl)biphenyl-3-yl)methanesulfonamide;N-(2-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)methanesulfonamide;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-methoxybenzonitrile;N-(4′-((trans)-2-aminocyclopropyl)biphenyl-2-yl)methanesulfonamide;4′-((trans)-2-aminocyclopropyl)-6-methoxybiphenyl-3-carbonitrile;N-(4′-((trans)-2-aminocyclopropyl)-6-methoxybiphenyl-3-yl)methanesulfonamide;4′-((trans)-2-aminocyclopropyl)-6-hydroxybiphenyl-3-carbonitrile;N-(4′-((trans)-2-aminocyclopropyl)-6-hydroxybiphenyl-3-yl)methanesulfonamide;3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-hydroxybenzonitrile;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-hydroxyphenyl)methane-sulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenyl)ethanesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenyl)methanesulfonamide;3-(6-((trans)-2-aminocyclopropyl)pyridin-3-yl)phenol;(Trans)-2-(5-(3-methoxyphenyl)pyridin-2-yl)cyclopropanamine;4-(6-((trans)-2-aminocyclopropyl)pyridin-3-yl)phenol;2-(6-((trans)-2-aminocyclopropyl)pyridin-3-yl)phenol;2-(5-((trans)-2-aminocyclopropyl)thiophen-2-yl)phenol;3-(5-((trans)-2-aminocyclopropyl)thiophen-2-yl)phenol;4-(5-((trans)-2-aminocyclopropyl)thiophen-2-yl)phenol;2-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenol;3-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenol;4-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenol;2-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;3-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;2-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;3-(2-((trans)-2-aminocyclopropyl)thiazol-5-yl)phenol;3-(5-((trans)-2-aminocyclopropyl)pyrimidin-2-yl)phenol;4-(5-((trans)-2-aminocyclopropyl)pyrimidin-2-yl)phenol;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-4-methoxyphenyl)methane-sulfonamide;N-(4′-((trans)-2-aminocyclopropyl)-5-chloro-[1,1′-biphenyl]-3-yl)methanesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-chlorophenyl)methanesulfonamide;N-(4′-((trans)-2-aminocyclopropyl)-4-fluoro-[1,1′-biphenyl]-3-yl)methanesulfonamide;N-(5-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-2-fluorophenyl)methanesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)ethanesulfonamide-;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)-4-cyanobenzenesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)-3-cyanobenzenesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)phenyl)-2-cyanobenzenesulfonamide;N-(3-(5-((trans)-2-aminocyclopropyl)pyridin-2-yl)-5-(trifluoromethyl)phenyl)-4-cyanobenzenesulfonamide;N-(4′-((trans)-2-aminocyclopropyl)-[1,1′-biphenyl]-3-yl)-1,1,1-trifluoromethanesulfonamide;4′-((trans)-2-aminocyclopropyl)-6-hydroxy-[1,1′-biphenyl]-3-carbonitrile;4′-((trans)-2-aminocyclopropyl)-[1,1′-biphenyl]-2-ol,4′-((trans)-2-aminocyclopropyl)-3′-methoxy-[1,1′-biphenyl]-3-ol;N-(3-(5-((trans)-2-aminocyclopropyl)thiazol-2-yl)phenyl)-2-cyanobenzenesulfonamide;or a pharmaceutically acceptable salt or solvate thereof.

In other embodiments, LSD1 inhibitor compounds are selected fromphenylcyclopropylamine derivatives, as described for example byOgasawara et al. (2013, Angew. Chem. Int. Ed. 52:8620-8624, which ishereby incorporated by reference herein in its entirety). Representativecompounds of this type are represented by formula XII:

wherein Ar₁ is a 5 to 7 membered aryl or heteroaryl ring;

Ar₂ and Ar₃ are each independently selected from a 5 to 7 membered arylor heteroaryl ring, optionally substituted with 1 to 3 substituents;

R₁ and R₂ are independently selected from hydrogen and hydroxyl or takentogether R₁ and R₂ form ═O, ═S or ═NR₃;

R₃ is selected from hydrogen, —C₁₋₆alkyl or —OH;

m is an integer from 1 to 5; and

n is an integer from 1 to 3;

or a pharmaceutically acceptable salt thereof.

In particular embodiments of formula (VII), one or more of the followingapplies:

Ar₁ is a six membered aryl or heteroaryl ring, especially phenyl,pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and1,2,3-triazine, more especially phenyl;

Ar₂ is a six membered aryl or heteroaryl ring, especially phenyl,pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and1,2,3-triazine, especially phenyl; especially where the six memberedaryl or heteroaryl ring is optionally substituted with one optionalsubstituent, especially in the 3 or 4 position;

Ar₃ is a six membered aryl or heteroaryl ring, especially phenyl,pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and1,2,3-triazine, especially phenyl;

especially where the six membered aryl or heteroaryl ring is optionallysubstituted with one optional substituent, especially in the 3 or 4position.

Particular optional substituents for Ar₁ and Ar₂ include —C₁₋₆alkyl,—C₂₋₆alkenyl, —CH₂F, —CHF₂, —CF₃, halo, aryl, heteroaryl,—C(O)NHC₁₋₆alkyl, —C(O)NHC₁₋₆alkylNH₂, —C(O)-heterocyclyl, especiallymethyl, ethyl, propyl, butyl, t-butyl, —CH₂F, —CHF₂, —CH₃, Cl, F,phenyl, —C(O)NH(CH₂)₁₋₄NH₂ and —C(O)-heterocyclyl;

R₁ and R₂ taken together form ═O, ═S or ═NR₃, especially ═O or ═S, moreespecially ═O;

R₃ is H, —C₁₋₃alkyl or —OH, especially H, —CH₃ or —OH.

m is 2 to 5, especially 3 to 5, more especially 4,

n is 1 or 2, especially 1.

In some embodiments the compounds of formula (XII) are compounds offormula (XIIa):

wherein Ar₂ and Ar₃ are as defined for formula (XII).

Non-limiting compounds represented by formula XII include the following:

Compound Ar₂ Ar₃ 1b phenyl phenyl 1c 4-methylphenyl phenyl 1d4-t-butylphenyl phenyl 1e 4-chlorophenyl phenyl 1f 4-fluorophenyl phenyl1g 4-phenyl-phenyl Phenyl 1h 4-trifluoromethylphenyl Phenyl 1i3-(2-aminoethylcarbamoyl)phenyl Phenyl 1j3-(piperazine-1-carbonyl)phenyl Phenyl 1k 4-phenyl-phenyl 4-methylphenyl1l 4-phenyl-phenyl 4-fluorophenyl 1m 4-phenyl-phenyl 4-phenyl-phenyl 1n4-phenyl-phenyl 4-t-butylphenyl 1o 4-phenyl-phenyl 3-methylphenyl 1p4-phenyl-phenyl 3-fluorophenyl 1q 4-phenyl-phenyl 3-phenyl-phenyl

The synthesis and inhibitory activity of the compounds of formula (VII)are described by Ogasawara et al. (2013, supra).

Other LSD1 inhibitors include, but are not limited to those, e.g.,disclosed in Ueda et al. (2009. J. Am. Chem. Soc. 131(48):17536-17537)including; Mimasu i (2010. Biochemistry June 22. [Epub ahead of print]PMID: 20568732 [PubMed—as supplied by publisher].

Other phenylcyclopropylamine derivatives and analogs are found, e.g., inKaiser et al. (1962, J. Med. Chem. 5:1243-1265); Zirkle et al. (1962. J.Med. Chem. 1265-1284; U.S. Pat. Nos. 3,365,458; 3,471,522; 3,532,749)and Bolesov et al. (1974. Zhumal Organicheskoi Khimii 10:8 1661-1669)and Russian Patent No. 230169 (19681030).

The invention not only encompasses known LSD (e.g., LSD1 or LSD2)inhibitors but LSD inhibitors identified by any suitable screeningassay. Accordingly, the present invention extends to methods ofscreening for modulatory agents that are useful for inhibiting a LSD(e.g., LSD1 or LSD2) and, in turn, for altering at least one of: (i)formation; (ii) proliferation; (iii) survival; (iv) viability; (v)maintenance; (vi) EMT; or (vii) MET of a LSD-overexpressing cell (e.g.,a CSC), or for treating or preventing a cancer (e.g., a metastaticcancer). In some embodiments, the screening methods comprise (1)contacting a preparation with a test agent, wherein the preparationcomprises (i) a polypeptide comprising an amino acid sequencecorresponding to at least a biologically active fragment of a LSD (e.g.,LSD1 or LSD2), or to a variant or derivative thereof; or (ii) apolynucleotide comprising a nucleotide sequence from which a transcriptof a LSD gene (e.g., LSD1 or LSD2) or portion thereof is producible, or(iii) a polynucleotide comprising at least a portion of a geneticsequence (e.g., a transcriptional element) that regulates the expressionof a LSD gene (e.g., LSD1 or LSD2), which is operably linked to areporter gene; and (2) detecting a change in the level or functionalactivity of the polypeptide, the polynucleotide or an expression productof the reporter gene, relative to a reference level or functionalactivity in the absence of the test agent. A detected reduction in thelevel and/or functional activity of the polypeptide, transcript ortranscript portion or an expression product of the reporter gene,relative to a normal or reference level and/or functional activity inthe absence of the test agent, indicates that the agent is useful foraltering at least one of: (i) formation; (ii) proliferation; (iii)survival; (iv) viability; (v) maintenance; (vi) EMT; or (vii) MET of aLSD-overexpressing cell (e.g., a CSC), or for treating or preventing thecancer. Suitably, this is confirmed by analyzing or determining whetherthe test agent alters at least one of: (i) formation; (ii)proliferation; (iii) survival; (iv) viability; (v) maintenance; (vi)EMT; or (vii) MET of a LSD-overexpressing cell, or treats or preventsthe cancer.

Modulators falling within the scope of the present invention includeinhibitors of the level or functional activity of a LSD (e.g., LSD1 orLSD2), including antagonistic antigen-binding molecules, and inhibitorpeptide fragments, antisense molecules, ribozymes, RNAi molecules andco-suppression molecules as well as polysaccharide andlipopolysaccharide inhibitors of a LSD (e.g., LSD1 or LSD2).

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 Dalton.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,desirably at least two of the functional chemical groups. The candidateagent often comprises cyclical carbon or heterocyclic structures oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analoguesor combinations thereof.

Small (non-peptide) molecule modulators of a LSD (e.g., LSD1 or LSD2)are particularly advantageous. In this regard, small molecules aredesirable because such molecules are more readily absorbed after oraladministration, have fewer potential antigenic determinants, or are morelikely to cross the cell membrane than larger, protein-basedpharmaceuticals. Small organic molecules may also have the ability togain entry into an appropriate cell and affect the expression of a gene(e.g., by interacting with the regulatory region or transcriptionfactors involved in gene expression); or affect the activity of a geneby inhibiting or enhancing the binding of accessory molecules.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogues.

Screening may also be directed to known pharmacologically activecompounds and chemical analogues thereof.

Screening for modulatory agents according to the invention can beachieved by any suitable method. For example, the method may includecontacting a cell expressing a polynucleotide corresponding to a genethat encodes a LSD (e.g., LSD1 or LSD2) with an agent suspected ofhaving the modulatory activity and screening for the modulation of thelevel or functional activity of the LSD (e.g., LSD1 or LSD2), or themodulation of the level of a transcript encoded by the polynucleotide,or the modulation of the activity or expression of a downstream cellulartarget of the polypeptide or of the transcript (hereafter referred to astarget molecules). Detecting such modulation can be achieved utilizingtechniques including, but not restricted to, ELISA, cell-based ELISA,inhibition ELISA, Western blots, immunoprecipitation, slot or dot blotassays, immunostaining, RIA, scintillation proximity assays, fluorescentimmunoassays using antigen-binding molecule conjugates or antigenconjugates of fluorescent substances such as fluorescein or rhodamine,Ouchterlony double diffusion analysis, immunoassays employing anavidin-biotin or a streptavidin-biotin detection system, and nucleicacid detection assays including reverse transcriptase polymerase chainreaction (RT-PCR).

It will be understood that a polynucleotide from which a LSD (e.g., LSD1or LSD2) is regulated or expressed may be naturally occurring in thecell which is the subject of testing or it may have been introduced intothe host cell for the purpose of testing. In addition, thenaturally-occurring or introduced polynucleotide may be constitutivelyexpressed—thereby providing a model useful in screening for agents whichdown-regulate expression of an encoded product of the sequence whereinthe down regulation can be at the nucleic acid or expression productlevel. Further, to the extent that a polynucleotide is introduced into acell, that polynucleotide may comprise the entire coding sequence thatcodes for the a LSD (e.g., LSD1 or LSD2) or it may comprise a portion ofthat coding sequence (e.g., the active site of the LSD) or a portionthat regulates expression of the corresponding gene that encodes the LSD(e.g., a LSD1 promoter or a LSD2 promoter). For example, the promoterthat is naturally associated with the polynucleotide may be introducedinto the cell that is the subject of testing. In this instance, whereonly the promoter is utilized, detecting modulation of the promoteractivity can be achieved, for example, by operably linking the promoterto a suitable reporter polynucleotide including, but not restricted to,green fluorescent protein (GFP), luciferase, β-galactosidase andcatecholamine acetyl transferase (CAT). Modulation of expression may bedetermined by measuring the activity associated with the reporterpolynucleotide.

These methods provide a mechanism for performing high throughputscreening of putative modulatory agents such as proteinaceous ornon-proteinaceous agents comprising synthetic, combinatorial, chemicaland natural libraries. These methods will also facilitate the detectionof agents which bind either the polynucleotide encoding the targetmolecule or which modulate the expression of an upstream molecule, whichsubsequently modulates the expression of the polynucleotide encoding thetarget molecule. Accordingly, these methods provide a mechanism ofdetecting agents that either directly or indirectly modulate theexpression or activity of a target molecule according to the invention.

In alternative embodiments, test agents are screened using commerciallyavailable assays, illustrative examples of which include EpiQuik HistoneDemethylase LSDI Inhibitor Screening Assay Kit (Epigentek Group,Brooklyn, N.Y.) or the LSDI Inhibitor Screening Assay Kit (CaymanChemical Company, Ann Arbor, Mich.).

Compounds may be further tested in the animal models to identify thosecompounds having the most potent in vivo effects. These molecules mayserve as “lead compounds” for the further development of pharmaceuticalsby, for example, subjecting the compounds to sequential modifications,molecular modeling, and other routine procedures employed in rationaldrug design.

3. Therapeutic and Prophylactic Uses

In accordance with the present invention, it is proposed that agentsthat inhibit LSD (e.g., LSD1 or LSD2) function are useful as actives foraltering at least one of: (i) formation; (ii) proliferation; (iii)survival, (iv) viability, or (v) maintenance of a LSD-overexpressingcell (e.g., a CSC or a non-CSC tumor cell); (vi) EMT of aLSD-overexpressing cell (e.g., a CSC); or (vii) MET of aLSD-overexpressing cell (e.g., a CSC), or for treating or preventing acancer (e.g., a metastatic cancer). Thus, LSD inhibitor compounds, inaccordance with the present invention, are useful, suitably inpharmaceutical compositions, for treating or preventing cancers,including metastatic cancers. As such the present invention contemplatespharmaceutical compositions for treating, preventing and/or relievingthe symptoms of a malignancy, particularly a metastatic cancer, whereinthe compositions comprise an effective amount of a LSD (e.g., LSD1and/or LSD2) inhibitor and a pharmaceutically acceptable carrier and/ordiluent.

Any LSD inhibitor can be used in the compositions and methods of thepresent invention, provided that the inhibitor is pharmaceuticallyactive. A “pharmaceutically active” LSD inhibitor is in a form thatresults in a reduction, impairment, abrogation or prevention in the (i)formation; (ii) proliferation; (iii) survival; (iv) viability; or (v)maintenance of a LSD-overexpressing cell (e.g., a CSC or non-CSC tumorcell); or (vi) EMT of a LSD-overexpressing cell (e.g., a CSC), and/or inthe enhancement of (vii) MET of a LSD-overexpressing cell (e.g., a CSC),and/or in the treatment and/or prevention of a malignancy, particularlya metastatic cancer, including the prevention of incurring a symptom,holding in check such symptoms or treating existing symptoms associatedwith the metastatic cancer, when administered to an individual in needthereof.

Modes of administration, amounts of LSD inhibitor administered, and LSDinhibitor formulations, for use in the methods of the present invention,are routine and within the skill of practitioners in the art. Whether amalignancy, particularly a metastatic cancer, has been treated isdetermined by measuring one or more diagnostic parameters indicative ofthe course of the disease, compared to a suitable control. In the caseof an animal experiment, a “suitable control” is an animal not treatedwith the LSD inhibitor, or treated with the pharmaceutical compositionwithout the LSD inhibitor. In the case of a human subject, a “suitablecontrol” may be the individual before treatment, or may be a human(e.g., an age-matched or similar control) treated with a placebo. Inaccordance with the present invention, the treatment of a metastaticcancer includes and encompasses without limitation: (1) impairing,abrogating, reducing, preventing, or arresting the development of, the(i) formation; (ii) proliferation; (iii) maintenance; or (iv) EMT of aLSD-overexpressing cell (e.g., a CSC), or enhancing MET of aLSD-overexpressing cell (e.g., a CSC), in a patient; (2) treating acancer (e.g., a metastatic cancer) in a subject; (3) preventing a cancer(e.g., a metastatic cancer) in a subject that has a predisposition tothe cancer but has not yet been diagnosed with the cancer and,accordingly, the treatment constitutes prophylactic treatment of thecancer; or (iii) causing regression of a cancer (e.g., a metastaticcancer).

The compositions and methods of the present invention are thus suitablefor treating an individual who has been diagnosed with a metastaticcancer, who is suspected of having a metastatic cancer, who is known tobe susceptible and who is considered likely to develop a metastaticcancer, or who is considered likely to develop a recurrence of apreviously treated metastatic cancer. The metastatic cancer may behormone receptor positive or hormone receptor negative. In someembodiments, the metastatic cancer is hormone receptor negative and isthus resistant to hormone or endocrine therapy. In some embodiments inwhich the cancer is breast cancer, the breast cancer (e.g., thenon-breast CMC tumor cells) is hormone receptor negative (e.g., estrogenreceptor (ER) negative and/or progesterone receptor (PR) negative).

In some embodiments, and dependent on the intended mode ofadministration, the LSD inhibitor-containing compositions will generallycontain about 0.000001% to 90%, about 0.0001% to 50%, or about 0.01% toabout 25%, by weight of LSD inhibitor, the remainder being suitablepharmaceutical carriers or diluents etc. The dosage of the LSD inhibitorcan depend on a variety of factors, such as mode of administration, thespecies of the affected subject, age, sex, weight and general healthcondition, and can be easily determined by a person of skill in the artusing standard protocols. The dosages will also take into considerationthe binding affinity of the LSD inhibitor to its target molecule, itsbioavailability and its in vivo and pharmacokinetic properties. In thisregard, precise amounts of the agents for administration can also dependon the judgment of the practitioner. In determining the effective amountof the agents to be administered in the treatment or prevention of ametastatic cancer, the physician or veterinarian may evaluate theprogression of the disease or condition over time. In any event, thoseof skill in the art may readily determine suitable dosages of the LSDinhibitor without undue experimentation. The dosage of the activesadministered to a patient should be sufficient to effect a beneficialresponse in the patient over time such as impairment, abrogation orprevention in the formation, proliferation, survival, viability ormaintenance of CMCs (e.g., breast CMCs) and/or non-CMC tumor cells, ininhibition of EMT of non-CMC tumor cells, in stimulating MET of CMCs(e.g., breast CMCs) and/or in the treatment and/or prevention of ametastatic cancer. The dosages may be administered at suitable intervalsto ameliorating the symptoms of the hematologic malignancy. Suchintervals can be ascertained using routine procedures known to personsof skill in the art and can vary depending on the type of active agentemployed and its formulation. For example, the interval may be daily,every other day, weekly, fortnightly, monthly, bimonthly, quarterly,half-yearly or yearly.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active agent, which are sufficient to maintainLSD-inhibitory effects. Usual patient dosages for systemicadministration range from 1-2000 mg/day, commonly from 1-250 mg/day, andtypically from 10-150 mg/day. Stated in terms of patient body weight,usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patientbody surface areas, usual dosages range from 0.5-1200 mg/m²/day,commonly from 0.5-150 mg/m²/day, typically from 5-100 mg/m²/day.

In accordance with the practice of the present invention, inhibition ofLSD (e.g., LSD1 and LSD2) by the LSD inhibitor will result in reducedformation, proliferation, survival, viability or maintenance of CSCs,which will in turn result in fewer non-CSC tumor cells differentiatingfrom the CSCs and in more effective treatment of non-CSC tumor cellswith an auxiliary cancer therapy or agent. Thus, the present inventionfurther contemplates administering the LSD inhibitor concurrently withat least one cancer therapy that inhibits the proliferation, survival orviability of non-CMC tumor cells. The LSD inhibitor may be usedtherapeutically after the cancer therapy or may be used before thetherapy is administered or together with the therapy. Accordingly, thepresent invention contemplates combination therapies, which employ a LSDinhibitor and concurrent administration of an cancer therapy,non-limiting examples of which include radiotherapy, surgery,chemotherapy, hormone abalation therapy, pro-apoptosis therapy andimmunotherapy.

3.1 Radiotherapy

Radiotherapies include radiation and waves that induce DNA damage forexample, γ-irradiation, X-rays, UV irradiation, microwaves, electronicemissions, radioisotopes, and the like. Therapy may be achieved byirradiating the localized tumor site with the above described forms ofradiations. It is most likely that all of these factors effect a broadrange of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

Non-limiting examples of radiotherapies include conformal external beamradiotherapy (50-100 Grey given as fractions over 4-8 weeks), eithersingle shot or fractionated, high dose rate brachytherapy, permanentinterstitial brachytherapy, systemic radio-isotopes (e.g., Strontium89). In some embodiments the radiotherapy may be administered incombination with a radiosensitizing agent. Illustrative examples ofradiosensitizing agents include but are not limited to efaproxiral,etanidazole, fluosol, misonidazole, nimorazole, temoporfin andtirapazamine.

3.2 Chemotherapy

Chemotherapeutic agents may be selected from any one or more of thefollowing categories:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulphan and nitrosoureas); antimetabolites (for exampleantifolates such as fluoropyridines like 5-fluorouracil and tegafur,raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea;anti-tumor antibiotics (for example anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like paclitaxel and docetaxel; and topoisomerase inhibitors (forexample epipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan and camptothecin);

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), UHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5α-reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-erbb2 antibody trastuzumab [Herceptin™] and theanti-erbb1 antibody cetuximab [C225]), farnesyl transferase inhibitors,MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinaseinhibitors, for example other inhibitors of the epidermal growth factorfamily (for example other EGFR family tyrosine kinase inhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033)), for example inhibitors of the platelet-derived growth factorfamily and for example inhibitors of the hepatocyte growth factorfamily;

(v) anti-angiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™],compounds such as those disclosed in International Patent ApplicationsWO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compoundsthat work by other mechanisms (for example linomide, inhibitors ofintegrin αvβ3 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO00/40529,WO 00/41669, WO01/92224, WO02/04434 and WO02/08213;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense; and

(viii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant GDEPT(gene-directed enzyme pro-drug therapy) approaches such as those usingcytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy.

3.3 Immunotherapy

Immunotherapy approaches, include for example ex-vivo and in-vivoapproaches to increase the immunogenicity of patient tumor cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumor cell lines and approaches usinganti-idiotypic antibodies. These approaches generally rely on the use ofimmune effector cells and molecules to target and destroy cancer cells.The immune effector may be, for example, an antibody specific for somemarker on the surface of a malignant cell. The antibody alone may serveas an effector of therapy or it may recruit other cells to actuallyfacilitate cell killing. The antibody also may be conjugated to a drugor toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin,pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a malignantcell target. Various effector cells include cytotoxic T cells and NKcells.

3.4 Other Therapies

Examples of other cancer therapies include phototherapy, cryotherapy,toxin therapy or pro-apoptosis therapy. One of skill in the art wouldknow that this list is not exhaustive of the types of treatmentmodalities available for cancer and other hyperplastic lesions.

It is well known that chemotherapy and radiation therapy target rapidlydividing cells and/or disrupt the cell cycle or cell division. Thesetreatments are offered as part of the treating several forms of cancer,aiming either at slowing their progression or reversing the symptoms ofdisease by means of a curative treatment. However, these cancertreatments may lead to an immunocompromised state and ensuing pathogenicinfections and thus the present invention also extends to combinationtherapies, which employ both a LSD inhibitor, a cancer therapy and ananti-infective agent that is effective against an infection thatdevelops or that has an increased risk of developing from animmunocompromised condition resulting from the cancer therapy. Theanti-infective drug is suitably selected from antimicrobials, whichinclude without limitation compounds that kill or inhibit the growth ofmicroorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc.and thus include antibiotics, amebicides, antifungals, antiprotozoals,antimalarials, antituberculotics and antivirals. Anti-infective drugsalso include within their scope anthelmintics and nematocides.Illustrative antibiotics include quinolones (e.g., amifloxacin,cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine,lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin,lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin,tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin;gemifloxacin; and garenoxacin), tetracyclines, glycylcyclines andoxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline,lymecycline, methacycline, minocycline, oxytetracycline, tetracycline,tigecycline; linezolide, eperozolid), glycopeptides, aminoglycosides(e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins,gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin,spectinomycin, streptomycin, tobramycin), β-lactams (e.g., imipenem,meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine,cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole,cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam,cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin,cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam,carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin,azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin,dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin,penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin,cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090,CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736,CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g.,azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin,rosaramicin, roxithromycin, troleandomycin), ketolides (e.g.,telithromycin, cethromycin), coumermycins, lincosamides (e.g.,clindamycin, lincomycin) and chloramphenicol.

Illustrative antivirals include abacavir sulfate, acyclovir sodium,amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate,didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium,ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine,nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin,rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate,stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, andzidovudine.

Non-limiting examples of amebicides or antiprotozoals includeatovaquone, chloroquine hydrochloride, chloroquine phosphate,metronidazole, metronidazole hydrochloride, and pentamidine isethionate.Anthelmintics can be at least one selected from mebendazole, pyrantelpamoate, albendazole, ivermectin and thiabendazole. Illustrativeantifungals can be selected from amphotericin B, amphotericin Bcholesteryl sulfate complex, amphotericin B lipid complex, amphotericinB liposomal, fluconazole, flucytosine, griseofulvin microsize,griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, andterbinafine hydrochloride. Non-limiting examples of antimalarialsinclude chloroquine hydrochloride, chloroquine phosphate, doxycycline,hydroxychloroquine sulfate, mefloquine hydrochloride, primaquinephosphate, pyrimethamine, and pyrimethamine with sulfadoxine.Antituberculotics include but are not restricted to clofazimine,cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide,rifabutin, rifampin, rifapentine, and streptomycin sulfate.

As noted above, the present invention encompasses co-administration ofan LSD inhibitor in concert with an additional agent. It will beunderstood that, in embodiments comprising administration of the LSDinhibitor with other agents, the dosages of the actives in thecombination may on their own comprise an effective amount and theadditional agent(s) may further augment the therapeutic or prophylacticbenefit to the patient. Alternatively, the LSD inhibitor and theadditional agent(s) may together comprise an effective amount forpreventing or treating the metastatic cancer. It will also be understoodthat effective amounts may be defined in the context of particulartreatment regimens, including, e.g., timing and number ofadministrations, modes of administrations, formulations, etc. In someembodiments, the LSD inhibitor and optionally the cancer therapy areadministered on a routine schedule. Alternatively, the cancer therapymay be administered as symptoms arise. A “routine schedule” as usedherein, refers to a predetermined designated period of time. The routineschedule may encompass periods of time which are identical or whichdiffer in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration of the LSDinhibitor on a daily basis, every two days, every three days, every fourdays, every five days, every six days, a weekly basis, a monthly basisor any set number of days or weeks there-between, every two months,three months, four months, five months, six months, seven months, eightmonths, nine months, ten months, eleven months, twelve months, etc.Alternatively, the predetermined routine schedule may involve concurrentadministration of the LSD inhibitor and the cancer therapy on a dailybasis for the first week, followed by a monthly basis for severalmonths, and then every three months after that. Any particularcombination would be covered by the routine schedule as long as it isdetermined ahead of time that the appropriate schedule involvesadministration on a certain day.

Additionally, the present invention provides pharmaceutical compositionsfor reducing or abrogating the proliferation, survival or viability ofCMCs cells and for preventing or treating malignancies, particularlymetastatic cancers, which comprise a LSD inhibitor and optionally acancer therapy agent useful for treating malignancies. The formulationsof the invention are administered in pharmaceutically acceptablesolutions, which may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic ingredients.Depending on the specific conditions being treated, the formulations maybe administered systemically or locally. Techniques for formulation andadministration may be found in “Remington's Pharmaceutical Sciences,”Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may,for example, include oral, rectal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. For injection, the active agents or drugs of theinvention may be formulated in aqueous solutions, suitably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

The drugs can be formulated readily using pharmaceutically acceptablecarriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the invention tobe formulated in dosage forms such as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated. These carriers may be selected from sugars,starches, cellulose and its derivatives, malt, gelatin, talc, calciumsulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline, andpyrogen-free water.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Such compositions may beprepared by any of the methods of pharmacy but all methods include thestep of bringing into association one or more drugs as described abovewith the carrier, which constitutes one or more necessary ingredients.In general, the pharmaceutical compositions of the present invention maybe manufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical which can be used orally include push-fit capsules madeof gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

Dosage forms of the drugs of the invention may also include injecting orimplanting controlled releasing devices designed specifically for thispurpose or other forms of implants modified to act additionally in thisfashion. Controlled release of an agent of the invention may be achievedby coating the same, for example, with hydrophobic polymers includingacrylic resins, waxes, higher aliphatic alcohols, polylactic andpolyglycolic acids and certain cellulose derivatives such ashydroxypropylmethyl cellulose. In addition, controlled release may beachieved by using other polymer matrices, liposomes or microspheres.

The drugs of the invention may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture (e.g., the concentration of an active agent,which achieves a half-maximal inhibition in activity of a LSDpolypeptide). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of such drugs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. The dataobtained from these cell culture assays and animal studies can be usedin formulating a range of dosage for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See for example Fingl et al., 1975, in“The Pharmacological Basis of Therapeutics”, Ch. 1 p1).

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a tissue, which is preferably subcutaneous or omental tissue, oftenin a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tissue-specific antibody.The liposomes will be targeted to and taken up selectively by thetissue.

In cases of local administration or selective uptake, the effectivelocal concentration of the agent may not be related to plasmaconcentration.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLES Example 1 CSCs and Epigenetic Regulation

CSCs have several important in vitro properties. First, breast CSCs arecharacterized by the key surface markers CD44^(high) CD24^(low) (FIG. 1A). Second, CSC-enriched populations have the ability to form sphericalcolonies in suspension cultures (termed mammospheres for breast CSCs)(FIG. 1 C). Third, CSC-enriched populations show enhanced resistance tochemotherapy and ionizing radiation. Fourth, they display a distincttranscriptome profile (FIG. 1 B). Therefore, CSCs represent a distinctpopulation of cancer cells with distinct molecular mechanismsmaintaining their unique in vitro properties.

Example 2 LSD1 Functions as an Epigenetic Regulator of Human Breast CSCs

H3K4me1 and LSD1 ChIP assays were conducted across the CD44 promoterregion in three human CSC models. (EC, MCF-7 epithelial cell line; BC,MDA-MB 231 basal cell line). The results presented in FIG. 2 show thatLSD1, H3K4me1 and H3K9me1 histone modification are enriched across keygenes whose transcripts are predominantly enriched in breast CSCs (e.g.,CD44, UPAR, laminin).

Example 3 Nuclear Staining of LSD1 in Human Normal and Breast CancerTissue

Nuclear staining of normal and human breast cancer tissue was carriedout using an anti-LSD1 antibody. The results presented in FIG. 3 reveal:that normal breast tissue shows strong nuclear immunoreactivity for LSD1(Abcam; 1/50 dilution) in normal ductal epithelium and adjacent stromalcells (see Panel A in FIG. 3); and that Grade 3 invasive ductalcarcinoma (ER/PR⁻Her2⁺) shows strong nuclear immunoreactivity for LSD1(see Panel B in FIG. 3). Inspection of a photomicrograph of normalbreast tissue (see Panel C in FIG. 3) shows patchy weak membranousstaining for CD44 (BD Pharmingen; 1/50 dilution) in normal ductalepithelium. By contrast, the same grade 3 invasive ductal carcinomashows strong circumferential membranous staining with an antibodytargeted against CD44 (see Panel D in FIG. 3).

Example 4 LSD1 siRNA Knockdown in Human Breast CSC Models Abolishes CSCs

The present inventors successfully knocked down LSD1 in all their CSCmodels (see, FIG. 1) using validated pooled siRNAs (Santa Cruz)according to published protocols. Greater than 85% knockdown of LSD1 wasobserved in these systems (FIG. 4A). LSD1 knockdown led to: a decreasein CSCs as measured by FACS in the Basal/Metastasis model (FIG. 4B) andMCF-IM model (FIG. 4C); decreased transcription of CSC marker genes suchas CD44 (FIG. 4D); reduction in mammospheres as measured by themammosphere assay (data not shown); decrease in LSD1 and H3K4me1 marksmeasured by chromatin immunoprecipitation (ChIP) (FIG. 4E); inhibitionof CD44 active chromatin domains measured by FAIRE (FIG. 4F).

Overall, these data show that LSD1 functions as an epigenetic regulatorof human breast CSCs.

Example 5 LSD2 siRNA Knockdown in Human Breast CSC Models Abolishes CSCs

The present inventors also successfully knocked down LSD2 in a breastCSC model using validated pooled siRNAs (Santa Cruz). Again, theyobserved >85% knockdown in CD44^(high) CD24^(low) breast cancer cells asassessed by flow cytometry (see, FIGS. 5A and B). LSD1 knockdown alsoled to: reduction in mammospheres as measured by the mammosphere assay(FIG. 5C).

Overall, these data show that LSD2 functions as an epigenetic regulatorof human breast CSCs.

Example 6 Inhibition of LSD1 by Specific Inhibitor Reduces CSC Formation

LSD1 specific inhibitor NCD-38 results in inhibition CSC formation inMCF-IM model as monitored by FACS (FIGS. 6A and B) and mammosphere assay(FIGS. 1C and D).

Example 7 Inhibition of LSD1 Results in Mesenchymal to EpithelialTransition (Met)

Treatment of basal metastatic model (MDA-MB 231) with LSD1 specificinhibitor NCD-38 results in a decrease of CSC formation (FIGS. 7A and B)and in conversion of mesenchymal cells in to epithelial cells (FIG. 7C).

Example 8 Reduction of Tumor Sizes by LSD1 Inhibitor in Combination withChemotherapy

LSD1 inhibitor pargyline results in reduced tumor size in combinationwith chemotherapeutic agent Docetaxel in mice xenograft model (FIG. 8A,B, FIG. 9A, B).

Example 9 Inhibition of CSC Subpopulation by Combination of Chemotherapyand LSD1 Inhibitor

Treatment of mice with LSD1 inhibitor, pargyline along withchemotherapeutic agent Docetaxel resulted in reduced CSC subpopulationas measured by FACS (FIG. 10A) and transcript (FIG. 10B).

Materials and Methods General Reagents

Chemicals

All the reagents used were either classified molecular biology oranalytical grade.

Cell Lines from the American Type Culture Collection (ATCC)

The adherent human mammary adenocarcinoma cell lines including MCF-7(ATCC® number HTB-22), MDA-MB-231 (ATCC® number HTB-26), MDA-MB-468(ATCC® number HTB-132), human adherent mammary ductal carcinoma T-47D(ATCC® number HTB-133) and human cervix carcinoma including HeLa (ATCC®number CCL-2) were obtained from ATCC (VA, USA). Stocks were stored at−196° C. in 5×10⁶ cells/mL aliquots in either RPMI or DMEM Completemedium (containing 45% heat inactivated foetal calf serum (FCS;Sigma-Aldrich, St. Louis, Mo.) and 9% dimethyl sulfoxide (DMSO) solution(Cambridge Isotope Laboratories, Inc., Andover, Mass.). Cell stocks werethawed in complete media, RPMI or DMEM, and checked for mycoplasmacontamination. Cell lines were frozen as stocks described in cryotubes(NUNC, Roskilde, Denmark) at −70° C. overnight and removed to long termstorage in liquid nitrogen.

Media, Buffers, and Solutions

All media, buffers and solutions were obtained either from JCSMRMedia/Wash-up Facility, ANU, Canberra, Australia or purchased from Gibco(Invitrogen Corporation, NY). RPMI-1640 (Gibco #11875-093) or Dulbecco'sModified Eagle Medium (DMEM) (1×, liquid, low glucose, Gibco #12320-32)complete cell culture media were freshly prepared for tissue cultureaccording to experimental demand by supplementing RPMI/DMEM plus HEPESwith 10% heat inactivated FCS, 0.1% PSN antibiotics and 2 mML-glutamine.

Antibiotics

Antibiotic stocks were dissolved in DDW filtered through 0.22 μm filters(Millipore, NSW, Australia) and prepared by the JCSMR Media Facility,ANU, Canberra, Australia. Penicillin, streptomycin and neomycin (PSN)antibiotics (1000× stock): 30.07 g/L Penicillin G Sodium (MPBiomedicals, LLC), 50 g/L Streptomycin Sulphate (Sigma-Aldrich, St.Louis, Mo.) and 50 g/L Neomycin Sulphate (Sigma-Aldrich, St. Louis,Mo.). PSN was added to all RPMI/DMEM Complete medium except whereotherwise stated.

Oligonucleotides

Primer/probe sets for gene expression analysis were purchased onlinefrom Taqman® Gene expression Assays (Applied Biosystems, Foster City,Calif.). Human TaqMan® probe sets used for quantitative cDNA real-timePCR included laminin-5, Fibronectin, Integrin-β, snail-1, uPAR,E-cadherin, vimentin, MMP-1, Zeb1, CD44, CD24, LSD1, PKC-θ andcyclophilin A. All primer sequences used for quantitative real-time PCRanalysis of transcript are listed in Table 3.

All genomic DNA oligonucleotides were purchased online as GuaranteedOligos from EasyOligoes Australia (Sigma-Aldrich, St. Louis, Mo.) as 100μM stocks. Primer concentrations were optimized to achieve similaramplification efficiencies between different primer sets for the samegene. All oligonucleotide primer sequences used for quantitativereal-time PCR are listed in Table 4.

Antibodies and Conjugates

All antibodies for this study were purchased from commercial sources.Details of all commercially purchased antibodies are listed in Table 5.

Kits

100 mM dNTP set (4×25 μmol) kit (Astral scientific Pty. Ltd., NSW,Australia)

Platinum® Taqman DNA Polymerase (Invitrogen, Carlsbad, Calif.)

QIAmp® Blood Mini Kit (Qiagen, Valencia, Calif.)

Superscript™ III RNaseH-Reverse Transcriptase kit (Invitrogen, Carlsbad,Calif.)

PowerSYBER Green PCR Master Mix (Applied Biosystems, Foster City,Calif.)

Taqman® universal PCR Master Mix (Applied Biosystems, Foster City,Calif.)

Taqman® MicroRNA Reverse Transcription kit

Enzymes and markers

Complete Protease Inhibitor Cocktail Tablets (Roche Diagnostics,Mannheim, Germany)

DNAse I, RNase-free (Roche Diagnostics, Mannheim, Germany)

Proteinase K (solution), RNA grade (Invitrogen, Carlsbad, Calif.)

Superscript™ III RNaseH-Reverse Transcriptase (Invitrogen, Carlsbad,Calif.)

Cell Culture

Mammalian Cell-Lines

The adherent human mammary adenocarcinoma MCF-7 cells lines were grownin DMEM Complete media. After thawing the cells were kept in DMEMComplete media in a sterile 75 cm² flask for 2 days before firstsplitting. Once confluent (after 2 days), cells were washed with 10 mLpre-warmed D-PBS (Gibco-BRL, Gaitherburg, Mass.) before adding 1 mL of0.05% trypsin-EDTA (1×) (Gibco-BRL, Gaitherburg, Mass.) on washed cells.Cells were then incubated for 3 minutes at 37° C. in order to detach thecells, followed by addition of another 10 mL DMEM media. Cells were thencentrifuged at 300×g for 10 minutes prior to re-suspension in 1 mL offresh complete DMEM media and counted subsequently by Vi-CELL-XR counterbefore splitting at desired density of cells. Cells were passagedsubsequently every 2-3 days depending on experimental demands and weresub-cultured when reached 80% confluences. For most of the experiments,4×10⁴ cells/well of a 24 well plate, 4×10⁵ cells/75 cm² flask or 4×10⁶cells/375 cm² flask were seeded one day before the experiment unlessotherwise stated. MDA-MB-231All cells were grown in a humidifiedatmosphere of 5% CO₂/O₂ and incubated at 37° C. in a Hepa-FilteredInfrared (IR) Incubator (Forma Scientific Inc., Materietta, Ohio). Allother cell lines used were grown and passaged in same way except usingRPMI-1640 Complete media.

TABLE 3 HUMAN PRIMER/PROBE SET FOR REAL-TIME PCR FROM TAQMAN ® NCBILocation Accession Amplicon size Gene Chromosome Assay details number(bp) CD44 Chr. 11 - 35160417-35253949 Hs00153304_m1 NM_000610.3 86 CD24Chr. Y - 21152526-21154705 Hs00273561_m1 NM_013230.2 162 uPAR Chr. 19 -44150248-44174502 Hs00182181_m1 NM_002659.2 64 NM_001005377.1NM_001005376.1 Zeb1 Chr. 10 - 31608101-31818742 Hs00611018_m1NM_001128128.2 77 NM_030751.4 LSD1 Chr. 1: 23345941-23410184Hs01002741_m1 NM_001009999.2 63 NM_015013.3 Laminin-5 Chr. 1 -183155174-183214262 Hs00194333_m1 NM_005562.2 132 NM_018891.2

TABLE 4 HUMAN PRIMER/PROBE SET FOR REAL-TIME PCR FROM EASYOLIGO GeneGenomic primer (5′→3′) CD44 OLIGO Seq FORWARD PRIMERTGAGCTCTCCCTCTTTCCAC REVERSE PRIMER TTGGATATCCTGGGAGAGGA uPAR OLIGO SeqFORWARD PRIMER GGGAAGCAAAGCAAGGGTTA REVERSE PRIMERGTTTTGTCAGGAGGGATACTGG

TABLE 5 ANTIBODY DETAILS Final Catalogue Stock concentration AntibodySupplier number concentration used H3K4Me2 Millipore 07-030 Polyclonal2.5 μg/tube Antibody H3K4Me1 Abcam ab8895 0.3 mg/mL 2.5 μg/tube LSD1Millipore 07-705 Polyclonal   5 μg/tube Antibody Pol-II (c-21) Abcamab817 1 mg/mL   5 μg/tube APC Mouse BD 559942 Polyclonal 1:100 of stockAnti-Human Pharmingen Antibody CD44 PE Mouse BD 555428 Polyclonal 1:100of stock Anti-Human Pharmingen Antibody CD24

Cell Viability and Density Counts

Cell viability was determined by using Vi-CELL-XR Counter (BeckmanCoulter Ireland Inc., Galway, Ireland). Commercially available Vi-CELLCounter reagent packs were purchased from Beckman Coulter Ireland Inc.,Galway, Ireland. A 50 μL aliquot of re-suspended cells (in 1 mL media)was diluted 1:10, in 450 μL media and loaded in the Vi-CELL counter cupto perform cell count and viability check. Cell viability counts wereconstantly >98% unless otherwise stated.

Mycoplasma Detection

Prior to freezing and after thawing cells were always checked formycoplasma contamination and only mycoplasma-free cells were used forall the experiments. Mycoplasma detection was performed with MycoAlert QMycoplasma detection kit (Lonza, Me. USA).

Stimulation Conditions

MCF-7 cells were seeded one day before stimulation at a set densityaccording to the experimental demand. Potential inducers of EMT weretested including Phorbol 12-Myristate 13-acetate (PMA) (Sigma-Aldrich,St. Louis, Mo.), and TGF β (R&D systems, Minneapolis, Minn.) at variousconcentrations and incubation length as specified in Table 6.

LSD1 Inhibition Conditions

The commercial LSD1 inhibitors used and supplier information aredescribed in Table 7. Corresponding control samples with equivalentconcentration of dissolving media or vehicle, usually DMSO (unlessotherwise stated) was also included for each experiment.

Epithelial to Mesenchymal Transition (EMT) Assay

EMT assays were performed in 24-well plates (Costar, Corning Inc.,Corning, N.Y., USA). For this assay, usually MCF-7 cells were seeded at4×10⁴/well/500 μL media one day before the experiment unless otherwisespecified. Cells were stimulated with various EMT stimuli prepared inwarm media the following day and monitored for EMT changes under themicroscope specified below. The percentage of EMT was generallycalculated based on phenotype counting 100 cells per field under themicroscope. All the potential EMT stimuli used herein are described inTable 6 and they were prepared and stored as per supplier specification.

TABLE 6 DETAILS OF POTENTIAL EMT STIMULI Final Supplier & Stockconcentration & EMT Inducer Catalogue number concentration stimulationtime PMA Sigma-Aldrich  1 mg/mL 20 ng/mL to (P8139) 0.2 ng/mL for 2 hourto 60 hour Recombinant R&D systems 10 μg/mL in PBS 2.5 ng/mL for humanTGF-β (240/b-CF) 2 hour to 60 hour

TABLE 7 LSD1 INHIBITOR INFORMATION Supplier & Final concentration &Inhibitor Catalogue number Pre-incubation time Pargyline Cayman  3 mMfor 17 hour (10007852) Phenelzine Sigma 500 μM for 17 hour (P6777)Tranylcypromine Enzo life sciences  1 μM for 17 hour (EL-217)

Mammosphere Assay

The following mammosphere culture media components were purchased fromStemCell Technologies Inc., BC, Canada: Mammocult Basal medium (Human)(catalogue number-05621), Mammocult Proliferation Supplements (Human)(catalogue number-05622), heparin (catalogue number-07904) andhydrocortisone (catalogue number-07904). Hydrocortisone powder wasfreshly dissolved into mammocult basal medium to get 10⁻⁴M solution onthe day of experiment. 50 mL of Mammocult complete media was thenprepared by addition of 45 mL Mammocult basal medium, 5 mL mammocultproliferation supplements, 100 μL of the 0.2% heparin stock, 500 μL of10⁻⁴M stock of hydrocortisone and 50 μL of the PNS. Mammocult completemedia was either used on same day or stored for not more than 7 days at4° C.

MCF-7 cells were grown in a 175 cm² cell culture flask and harvestedusing a cell scraper (Zellschaber, Switzerland) or FACS sorted asspecified below. Importantly trypsin treatment was never used forharvesting cells as it interferes with the mammosphere assay. Harvestedcells were then re-suspended in 1 mL mammocult complete media andcentrifuged at 500×g for 3 minutes at 20° C. The cell pellet was thenre-suspended in 1 mL mammocult complete media before counting the cellson the Vi-CELL counter. Cell dilutions were then prepared to stain40,000 cells/2 mL and 2 mL of cells were seeded in the 6 well-ultra lowadherent, flat bottom plates (Costar, Corning Inc, Corning, N.Y., USA).Cells were either stimulated or not treated according to theexperimental protocol and incubated at 37° C., under 5% CO₂ for 7 daysin a Hepa-Filtered Infrared (IR) Incubator (Forma Scientific Inc.,Materietta, Ohio). Mammospheres larger than 60 μm were counted per wellon day 7 and pictures were taken with an Olympus microscope. All themammosphere assays were performed in duplicate wells and the entireprocedure was repeated at least twice.

Immunofluorescence

Fluorescence-Activated Cell Sorting (FACS)

Cells from each well/flask were harvested by means of trypsin treatmentfollowed by two washes with PBS. Washed cells then re-suspended in anantibody cocktail consisting anti-CD44-APC, anti-CD24-PE antibodies(details in Table 5) and Hoechst 33258 dye (final dilution of 1:1000)(Invitrogen, Carlsbad, Calif.) in 1% FCS-PBS solution. Cellsre-suspended in antibody cocktail were incubated for 20 minutes at 4° C.Cells were next washed twice with PBS and re-suspended in 1% FCS-PBSsolution (volume based on cell number) and kept on ice until analyzed byFACS. Forward scatter (FSC) and side scatter parameters were selectedwith FITC fluorochrome excited by 488 nm argonion laser, PE fluorochromeexcited by 488 nm and Hoechst fluorochrome excited by 350 nmhelium-cadmium UV laser.

Flow Cytometry data was produced using either BD FACS LSR Flow Cytometer(Becton Dickinson Biosciences) or BD FACS Aria™ II Flow Cytometer(Becton Dickinson Biosciences) and analyzed using the data acquisitionsoftware CellQuest Pro (Becton Dickinson Biosciences) and FlowJo (TreeStar Inc., Ashland, Oreg.) software at the MCRF facility, JCSMR, ANU,Canberra, Australia. Single colour controls were used to setcompensation parameters. Isotype controls were used for allcorresponding primary antibodies in each experiment.

CD44 and CD24 Staining Optimization in MCF-7 Cells

The FACS gating strategy used in this thesis is adopted and modifiedfrom the pioneer breast cancer stem cell publications (Al-Hajj et al.,2003) which sorted cells based on the CD44^(high) and CD24^(low)expression. Expression of CD44^(high) and CD24^(low) has been shown tobe associated with human breast cancer stem cells (Al-Hajj et al., 2003;Sleeman et al., 2006; Mani. et al., 2008). To confirm that anti-CD44-APCand anti-CD24-PE antibodies were specific, isotype control antibodieswere used. The isotype (negative controls) used for anti-CD24-PE was PEMouse-Anti-human IgG2aK and for anti-CD44-APC was APC-Mouse-Anti-humanIgG2bK. First, all the cells were stained with Hoechst 33258 to monitorcell viability. In addition cells were stained with varyingconcentrations of either APC or PE isotypes controls.

Microscopy

Fluorescence Microscopy

Cells were stained as outlined above and mounted on coverslips. Stainedcells were viewed under oil immersion at ×100 magnification usingOlympus Fluorescence 1×71 microscope (Olympus, Tokyo, Japan) or 60×magnification or Leica confocal microscope (Leica microsystems). Imageson Olympus Fluorescence 1×71 microscope were captures using DPControllercamera software version 1.2.1.108 (2002 Olympus optical Co., LTD) andimages on Leica confocal microscope were captured using Leicaapplication suite, 2.0.0 program. Images were analyzed using PhotoshopCS3 (Adobe Systems Inv., San Jose, Calif.). GFP/FAM vector transfectedwells or flasks were viewed under Olympus Fluorescence 1×71 microscopeusing FITC excitation filter 406-495 nM filter (WIB).

Phase-Contrast Microscopy

Phase contrast microscopy was utilized for EMT and wound healing assaysunder 10× or 20× magnification of Olympus Fluorescence 1×71 microscope.Images were captured and analyzed as described above except that woundhealing assay pictures were also analyzed by Image J software (Freesoftware in Public domain developed by NIH).

Transfection

DNA Transfection

Conditions were optimized for DNA transfection in MCF-7 and T-47D cellsby using commercially available transfection agents, FuGENE 6 (RocheDiagnostics, Mannheim, Germany) (the detailed method for FuGENE 6 isdescribed below). To achieve the maximum transfection efficiency,initially, the transfection reagents were examined at varying ratio ofreagent: DNA/oligo (GFP-expression vector for optimization). For all theDNA transfections cells were seeded at 1×10⁵ cells per well in 500 μL ofantibiotic-free media 24 hour prior to the commencement of transfectionin 24 well plate and transfections were performed as per themanufacture's guidelines. The dilution medium used for transfections,was OptiMEM® I Reduced-Serum Medium (1×), liquid (Invitrogen, Carlsbad,Calif.). Transfection percentage was checked after 36-48 hours by FACSas described above.

DNA Transfection Using FuGENE 6 Reagent

For a single reaction in 500 μL total volume (per well of 24 wellplate), 0.9 μL FuGENE 6 was added into a 1.5 mL Eppendorf tubecontaining 10 μL of OptiMEM® I Reduced-Serum Medium, mixed well andincubated for 5 minutes at room temperature. After 5 minutes, either 0.4μg or 0.3 μg GFP tagged DNA (volume of DNA was calculated depending uponthe concentration of specific DNA oligonucleotide used) was added intothe tube to get a 2.25:1 or 3:1 ratio of FuGENE 6 respectively. DNA andthe complex were then incubated for 45 minutes at room temperature.After 45 minutes, 20 μL of the FuGENE-DNA complex was added on top ofthe cells drop wise and mixed by swirling the plate and plates wereincubated at 37° C. for 36-48 hours.

siRNA Transfection

Lyophilized Silencer® FITC Conjugate negative control siRNA (Mock)(sc-36869); validated LSD1 siRNA (sc-60970) and LSD2 siRNA (sc-95467)was purchased from Santa Cruz Biotechnologies, California. Thespecificity of these siRNAs has been previously published (Sutcliffe etal., 2011. Molecular Cell 6:704-719; Yang et al., 2010. Proc. Natl.Acad. Sci. USA 107: 21499-21504. Forward transfections with 10 nM siRNAwere performed in MCF-7 cells by using Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.). Forward transfection methods were performed in 24well plates (4×10⁴ cells) for EMT assays and for transcript analysis,while for Chromatin Immuno-Precipitation (ChIP) assays where largequantities of cells were required, transfections were performed in 25cm² flasks (4×10⁵ cells).

Briefly, for setting up forward transfection reactions in 24 wellplates, 4×10⁴ MCF-7 cells per well were seeded in 500 μL of antibioticfree media 24 hours prior to transfection. 250 μL DEPC-water was addedin the 20 nmol lyophilized siRNA stock to get a 20 μM concentrationstock. To achieve a final siRNA concentration of 10 nM (for one reactionin total volume of 500 μL media in one well of 24 well plate), 3 μL of20 μM siRNA stock was further diluted in 50 μL Opti-MEM® I and incubatedfor 5 minutes at room temperature. This step was immediately followed bya further dilution of Lipofectamine by adding 1 μL Lipofectamine (forone reaction) in 50 μL Opti-MEM® I, followed by 5 minutes incubation atroom temperature. 50 μL of diluted siRNA and 50 μL of dilutedLipofectamine solutions were then mixed together to get 100 μL of thesiRNA-Lipofectamine complex. This complex was subsequently incubated inthe dark, at room temperature for 20 minutes. The resultantsiRNA-Lipofectamine complex was carefully pipette onto the surface ofthe cells and mixed by gently rocking the plate back and forth. Plateswere incubated for 48-72 hours and transfection efficiency was checkedby flow cytometry (refer above for detailed method of flow cytometryanalysis). The knockdown was checked with three methods: (1) transcriptanalysis on gene of interest was carried out to confirm the knockdown ofspecific gene, for example, LSD1 knockdown by LSD1 directed siRNA usingLSD1 oligonucleotide (described in Table 3) for real-time PCR fromTaqMan. (2) at the ChIP level (please refer to ChIP results for specificgenes) (3) optimization experiments were carried out for each siRNA toconfirm maximal transfection efficiency (70-80%) in MCF-7. Therefore,the results presented herein are reproducible. Each mock (control) andgene specific siRNA knockdown experiment was performed three independenttimes and only one representative experiment is shown in this section.

Molecular Biology Techniques

For RNA isolation procedure, all the pipettes, tube holders and gloveswere always pre-treated with RNase Zap (Ambion, VIC, Australia). RNAse,DNAse, pyrogen free, sterile microtubes (Axygen Scientific, Inc., UnionCity) and Pre-sterile aerosol resistant tips and fresh bench coat wereroutinely used for all the molecular biology work described in thissection.

Total RNA Isolation

For most of the experiments, total RNA was extracted from 5×10⁵ to 1×10⁶MCF-7 cells unless otherwise specified. Cells were first thawed in 1 mLof Trizol® Reagent (Invitrogen, Carlsbad, Calif.) for 5 minutes at roomtemperature to inactivate RNases, followed by trituration to dislodgethe cell pellet. The dissociated cells suspended in Tizol (1 mL) weretransferred into a 1.5 mL Eppendorf tube for 5 minutes forhomogenization. RNA was then extracted by addition of 200 μL chloroformand mixing was done vigorously before centrifuging the samples at8,000×g for 30 minutes at 4° C. The aqueous layer was then collectedinto a fresh 1.5 mL Eppendorf tube and an equal volume of isopropanololwas added to the aqueous layer and mixed gently. After 5 minutes at roomtemperature, samples were either snap frozen on dry ice and stored at−70° C. overnight or until the isolation procedure could bere-commenced. After thawing the samples quickly, the samples were againcentrifuged at 8,000×g for 30 minutes at 4° C. to precipitate the RNA.To remove all traces of isopropanolol, next the RNA pellets were washedwith 1 mL of ice cold 80% ethanol (Analytical UNIVAR, Seattle, Wash.)before centrifuging at 3,600×g for 10 minutes at 4° C. All ethanol thenwas removed and pellets were allowed to air dry for 5 minutes. RNAsamples were then solubilized by re-suspending them in 50 μL ofnuclease-free DEPC (Diethylpyrocarbonate treated) water (Ambion, VIC,Australia). Next, 2 μL of the sample was taken out to measure RNAquality and quantity on Nano-Drop® Spectrophotometer ND 1000 (NanodropTechnologies, Inc., Wilmington, Del., USA) using ND-1000 V3.30 software.All the RNA samples were found pure as they had A₂₆₀/A₂₈₀ ratio of1.9-2.1 and this ratio provides an estimate of RNA purity with respectto contaminants such as proteins that absorb in the UV spectrum.

First Stand cDNA Synthesis

Superscript™® III kit (Invitrogen, Carlsbad, Calif.) was used for cDNAsynthesis. Master mix-1 was prepared by adding 1 μL of 5 μM oligo (dT)and 1 μL of 100 mM dNTPs and mixed by flicking. Mastermix-2 was preparedby adding 2 μL of RT buffer, 2 μL of 3 mM mgCl₂, 2 μL of DTT mix, 1 μLof RNAse out, 1 μL of superscript III and mixed by flicking. For 1 μgRNA, 2 μL of the master mix-1 was added to the samples, mixed andincubated at 65° C. for 5 minutes and then samples were placed on ice tostop the reaction. This step was followed by addition of 10 μL of themaster mix-2 per sample and incubation of samples at 50° C. for 50minutes. The reaction was then stopped by incubating samples at 85° C.for 5 minutes followed by placing the samples on the ice for 2 minutes.Finally 1 μL of RNaseH was added to each sample and samples wereincubated at 37° C. for 20 minutes. All the samples were either snapfrozen on dry ice or used immediately for quantitative Real-Time PCRanalysis.

Quantitative Real-Time PCR (qRT-PCR) Analysis

TaqMan® Gene expression Assays (Applied Biosystems, Foster City, Calif.)were used to perform qRT-PCR on an ABI PRISM 7900 HT fast Real-Time PCRsequence detector (PerkinElmer/PE, Applied Biosystems, Foster City,Calif.) using the FAM probe channel. A total reaction volume of 10 μLwas used with cDNA diluted at 1:20 with DEPC water for the PCR, asdetailed in the manufacture's guidelines (PerkinElmer/PE, AppliedBiosystems, protocol PN 4333458). For all genomic DNA, Power SYBR Greenreal-time PCR (PerkinElmer/PE, Applied Biosystems, Foster City, Calif.)reactions were performed and the ChIP samples were diluted at 1:5. EachPCR was performed in duplicate wells using thermocycler conditions asfollows: stage 1-50° C. for 2 minutes for 1 cycle; stage 2-95° C. for 10minutes for 1 cycle; stage 3-95° C. for 15 seconds and 60° C. for 1minute for 40 cycles. For all the primers sets, no template controlswere always included to test for PCR amplification of any contaminatingDNA within the PCR mix. Dissociation curves were performed for eachprimer set to confirm amplification of a single product using thefollowing PCR conditions: stage 1-95° C. for 15 seconds; stage 2-60° C.for 20 seconds; with a minimum ramp speed to reach stage 3-95° C. for 15seconds. PCR reactions were performed using Optical PCR 384 wellreaction plates (Applied Biosystems, Foster City, Calif.).

Data Analysis of cDNA Experiments

All the threshold cycle (C_(t)) values from the PCR amplification plotswere converted to arbitrary copy number using the formula100000/2^(C_(t)−17) in Microsoft excel spread sheet, where a C_(t) valueof 17 was set to 10⁵ copies and assuming that each cycle increaseequated to a 2 fold increase in input DNA. All the primers were checkedagainst an amplicon standard curve to show that above formula producedresults that were similar to results obtained with amplificationstandard curve method. Cyclophilin A primer (section 0) PCR reactionswere performed concurrently for each experiment to normalize fordifferences in RNA input and cDNA synthesis. All experiments wereperformed in duplicate.

cDNA Synthesis for MicroRNA

The TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems,Foster City, Calif.) was used to convert total RNA into cDNA formicroRNA analysis with specific TaqMan® miRNA primers assays. Reagentsof TaqMan® MicroRNA Reverse Transcription Kit were allowed thawingcompletely on ice before preparing RT master mix by adding followingcomponents per 15 μL reaction volume; 0.15 μL dNTP mix (100 mM), 1 μLMultiscribe™ RT enzyme (50 U/μL), 1.5 μL 10×RT Buffer, 0.19 μL RNaseInhibitor (20 U/μL) and 4.16 μL Nuclease free water to get a totalvolume of 7 μL. All the components were mixed gently, centrifugedbriefly and then this RT master mix was placed on ice until the RTreaction plate was prepared. RT reaction plate was then prepared; byfirst combining total RNA (1-10 ng per 15 μL reaction) with the RTmaster mix in a ratio of 5 μL RNA:7 μL RT master mix. MicroRNA RTprimers were then thawed on ice and tubes to be used for RT reactionwere labeled with appropriate numbers. Next, 12 μL of the RT master mixcontaining total RNA was dispensed into each tube before adding 3 μL of5×RT primers to the appropriate tubes to bring the total volume 15 μLper tube. All the tubes were then mixed gently, centrifuged briefly andincubated on ice for 5 minutes or until ready to load on thermal cyclerfor reverse transcription at following thermal cycles at 15 μL reactionvolume—Hold for 16° C. for 30 minutes for 1 cycle; Hold for 42° C. for30 minutes for cycle 2; Hold for 85° C. for 5 minutes for cycle 3 and 4°C. for ∞ for cycle 4. After completion of the run, samples were eithersaved at −20° C. or used immediately for qPCR.

qPCR Amplification for MicroRNA cDNA

qPCR reaction was performed by preparing the 20 μL qPCR reaction mix byaddition of following components into appropriate tubes: 1 μL of TaqMan®small RNA Assay (20×); 1.33 μL of product from RT reaction; 10 μL ofTaqMan® Universal PCR master Mix II (2×, no UNG) and 7.67 μL nucleasefree water. Components were then mixed gently and centrifuged briefly.All the qPCR reactions were performed in triplicate. 20 μL of thecomplete qPCR reaction mix (including assay and RT product) were thentransferred into each of three wells of a 384-well plate. Plate was thensealed, centrifuged briefly and PCR amplification was performed asdescribed in section 2.5.2.

Chromatin Assays

Chromatin Immunoprecipitation (ChIP) Assay

Between 1-5×10⁶ Cells were harvested following various treatmentsaccording to the experimental requirement and re-suspended in 10 mL DMEMcompleter media at room temperature after counting then at Vi-CELLcounter. Cells were then cross linked with freshly prepared 1%paraformaldehyde (PFA) (Analytical UNIVAR, Seattle, Wash.) for 10minutes at room temperature with continuous but slow rotations on rotarywheel. Next, the reaction was quenched by the addition of 2M glycinesolution (AnalaR, Merck, Darmstadt, Germany) to a get a finalconcentration of 125 mM and mixed further for 10 minutes at roomtemperature on the rotary wheel. Cells were then washed three times with10 mL ice cold PBS and the cell pellet was either snap frozen on dry iceor used immediately afterwards. SDS Lysis Buffer (Upstate Biotechnology,Billerica, Mass.) was prepared by addition of 1× complete proteaseinhibitor solution (1 tablet dissolved in 1 mL DEPC water) (RocheDiagnostics, Mannheim, Germany) and cell pellet was then re-suspended in250 μL of the in the SDS lysis buffer for 10 minutes at roomtemperature. Cells were sonicated (10 sec pulses for 2 minutes on 1liter ice cold water mixed with ice, 70% maximum output) to shear thechromatin to obtain an average DNA fragment size of 250-500 bp using aCole Palmer Ultrasonic processor (Cole Plamer, Vernon Hills, Ill.).After the sonication, samples were centrifuged at 10,2000×g for 5minutes at room temperature to clear cellular debris and the supernatantwas then diluted to 1:10 with ChIP dilution buffer (UpstateBiotechnology, Billerica, Mass.). Antibodies as per the experimentrequirement were aliquoted to the 1.5 mL Eppendorf tubes before addingsonicated chromatin from 0.5-1×10⁶ cells diluted in the dilution bufferand the ChIP mixture was then incubated with antibodies overnight at 4°C. on rotary wheel. For all the experiments total genomic DNA withoutany antibody (named Total Inputs) for each condition was snap frozen andstored at −70° C., also a sample without any antibody (named Noantibody) was processed in parallel with the ChIP samples. Next, immunecomplexes were bound by addition of 60 μL of salmon sperm DNA/Protein Aagarose beads at 4° C. for 1 hour at rotary wheel. Samples were thencentrifuged at 2500×g for 2 minutes at 4° C. and the supernatant wasdiscarded before washing beads at 4° C. for 5 minutes on a rotary wheelwith each of the following washing buffers from Upstate Biotechnology(Billerica, Mass.) in the same order as described; first wash—500 μL oflow salt immune complex wash buffer; second wash—500 μL of high saltimmune complex wash buffer; third wash-500 μL of LiCi immune complexwash buffer; fourth wash-500 μL of low salt immune complex wash bufferand fourth wash—1 mL TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA).Protease Inhibitor-complete, 1× (Roche Diagnostics, Mannheim, Germany)was added to all the wash buffers immediately before use. DNA-proteincomplexes were then eluted from the beads with 400 μL of the freshlymade elution buffer (1% (w/v) SDS, 100 mM NaHCo₃) for 30 minutes at roomtemperature on rotary wheel. Samples and total input controls were thenincubated to hydrolyze cross links (or reverse-cross link) at 66° C.overnight after adding 16 μL of 5M sodium Chloride (Sigma-Aldrich, St.Louis, Mo.). Next day, samples were treated with 1 μL of Protease Ksolution (20 μg/μL) (Invitrogen, Carlsbad, Calif.) for 1 hour at 45° C.Digested protein was removed from the ChIP samples by addition of equalvolume of phenol-chloroform-isoamyl alcohol (25:24:1) saturated with 10mM Tris, pH 8.0, 1 mM EDTA (Sigma-Aldrich, St. Louis, Mo.), mixing thesamples and subsequent centrifugation at 10,200×g for 20 minutes at roomtemperature for collection of the aqueous layer. Genomic DNA wasprecipitated from aqueous fraction by the addition of 2.5 volumes ofice-cold absolute ethanol (Analytical UNIVAR, Seattle, Wash.), 0.1volume of 3 M sodium acetate buffer solution pH 5.2 (Sigma-Aldrich, St.Louis, Mo.) and 25 μg of GeneElute™ linear polyacrylamide(Sigma-Aldrich, St. Louis, Mo.) for at least 24 hours at −20° C. Next,samples were pelleted by centrifugation and washing with 80% ice coldethanol and pellets were allowed to air dry prior to suspension in 20 μLof the DEPC water for real time PCR analysis. The oligonucleotides usedfor performing real-time PCR on ChIP samples have been listed in Table4.

Sequential ChIP

Primary ChIP of was performed as described above until the TE bufferwashing step. Thus, immunoprecipitates from the primary ChIP weredissolved in 60 μL elution buffer containing 10 mM DTT (Superscript™ IIIRNaseH-Reverse Transcriptase kit (Invitrogen, Carlsbad, Calif.) in DEPCwater and incubated for 1 hour in a 37° C. water bath. Tubes wereflicked every 15 minutes during this incubation period. Next, sampleswere diluted with 1:40 of ChIP dilution buffer before taking 400 μL ofthe samples to be frozen at −70° C. to use as a total genomic input forsecondary ChIP. The second antibody was added to these samples andimmunoprecipitation was carried out again as the ChIP protocol (asdefined above.) except that immune complexes were bound to 60 μL of thesalmon sperm DNA/Protein A agarose beads for 2 hour at 4° C. SequentialChIP samples were then eluted, the cross-linking was reversed and thegenomic DNA was precipitated as method described in above. SequentialChIP analysis was carried out using the ChIP enrichment ratio methoddescribed below and then expressed as a fold change with respect to thenon-stimulated samples.

Data Analysis for ChIP Experiments

All ChIP assays were carried out in the presence of a non-antibodycontrol as well as an isotype specific control antibody. The negativecontrol is a non-antibody control and the enrichment values from theseare routinely low and are included as background subtractions in thecalculations of ChIP enrichment ratio. C_(t) values from the real timePCR amplification plots were first converted to arbitrary copy numberusing formula the 100000/2^(C_(t)−17). Sample data were normalized tothe corresponding total input arbitrary copy number. Fold change abovethe no antibody control was then calculated to get ChIP enrichmentratio. ChIP enrichment ratio values were multiplied by a factor of 10except the ChIP enrichment ratio values for histone modifications. Thismethod of analysis was adopted from Pokholok et al. (2006. Science 313:533-536) who established that the ChIP enrichment ratio presented on thelinear scale better emphasizes signal versus noise in the display ofChIP-on-ChIP data over a logarithmic scale (Pokholok et al., supra).Consequently, all the ChIP data in this thesis is presented on a linearscale. All chip assays represent mean±standard error (SE) of threeindependent experiments and analyzed in duplicate by real-time PCR. Insome cases fold change was calculated with respect to the non-stimulatedsample, which was set as 1. Statistical significance was determined bytwo-tailed Paired-t test using GraphPad Prism 5.03 for Windows.

All the chip samples were always normalized with the Total input (TI) ofeach corresponding sample. If the recovery of the ChIP samples was lowin the stimulated cells and CSC subset, then the arbitrary copiesachieved in the real-time PCR analysis would be low but this was not thecase and arbitrary copies from the samples were in same Ct range as theothers. Since the “ChIP enrichment ratio” method has been used foranalysis, of the recoveries of the ChIP DNA across the different samplesdid not affect the analysis and interpretation of the data. This ChIPanalysis method by using “ChIP enrichment ratio” is considered as thebest method for calculating ChIP data (Pokholok et al., supra).Therefore, the results were not likely to be effected due to poor ChIPrecoveries.

For sequential ChIP analysis, the genomic DNA recovered from thesequential ChIP experiments was quantified by SYBR Green real-time PCRusing primers specific for the promoter regions of the uPAR or miR 200c.The C_(t) values from the PCR amplification plots were converted toarbitrary copy number using the formula 100000/2^(C_(t)−17). The noantibody control was subtracted from the data for each sample, whichwere then normalized to the corresponding total input (TI-1) that wastaken prior to the first immunoprecipitation. Data generated were thennormalized to their respective 2^(nd) total immunoprecipitation input(TI-2). Finally, fold change was then calculated with respect to thenon-stimulated sample, which was set as 1. These values were then usedto prepare the sequential ChIP plots shown in Figures. This method ofsequential ChIP analysis has been adopted from Sutcliffe et al., 2009.Statistical significance was determined by two-tailed Paired-t testusing GraphPad Prism 5.03 for Windows. This method of sequential ChIPanalysis has been adopted from Sutcliffe et al., 2009.

In Vivo Murine Xenograft Model

To monitor the effect of LSD1 inhibitors in vivo on tumor recurrence, invivo murine xenograft model of breast cancer recurrence was used. TheMDA-MB 231 cell line model, which is one of the most robust andwell-established models was used in 6 weeks old BALB/c female nude mice.5×10⁶ MDA MB 231 cells were injected in the mammary fat pad of eachmouse. Mice were observed for the tumor appearance and growth. Once thetumors reach 50 mm³ volume size (determined by caliper measurement),treatments were initiated. Mice were monitored until tumors reach 500mm³ and sacrificed for collection of tumors. Each treatment groupconsisted 14 mice each (5 mice for tumor growth curve+3 mice each fortumor collection at 3 time points-treatment start day-week 0,chemotherapy tumor reduction-approx. week 4 and until tumor reach 500mm³ approx. week 7). Tumor volumes were measured every week and threemice from each group were sacrificed for IHC, FACS, and RNA extractionat above mentioned three time points.

Generation of Tumors

1. Cells were thawed from lot labeled MDA-MB-231 (Sep. 2, 2014).

2. Cells were expanded to 54×150 cm² flasks.

3. Cell suspension was then spun down at 1500 rpm for 5 min. at roomtemperature.

4. Supernatant were then discarded and cell pellet resuspended in 2.5 mLof cold PBS and 2.5 mL cold Matrigel™ (NOTE: Matrigel is in liquid stateonly when kept on ice).

5. Mice were then injected with 50 μL of the Matrigel™+5×10⁶ cellsuspension in PBS using 26″ needles and isoflurane as an anesthetic.Each 50 μL of the mixture contains 2×10⁶ MDA-MB-231 cells.

6. Mice were monitored and weighed and monitored daily till 15 May 2014.

7. Measurements for tumor volume began at day 7 post injection ofMDA-MB-231. Then the tumor volumes were measured daily till 15 May 2014.

Treatment of mice started at day 16 post injection of MDA-MB-231, wheretumors roughly reached a volume of 50 mm³.

Treatment of Mice

Group A: Control—20 μl of DMSO

Group B: 4 mg/kg of docetaxel (11 mice)

Group C: 100 mg/kg of pargyline (11 mice)

Group D: 4 mg/kg of docetaxel+100 mg/kg of pargyline (11 mice)

Collection of Samples

Tumor, spleen, liver, lungs and kidneys were collected into (1) fixativeagent for IHC and (2) 2 different RNase free 1.5 ml Eppendorf tubes tobe frozen down in the −80 freezer at Day 0 (1 mouse/group A-C).

At week four post treatment, tumor samples were collected (3 mice/group;taking the largest, smallest and average sized tumor) for flow cytometrywith CD44, CD24 and Hoechst staining. 1 ml from each single cellsuspension from each sample was spun down and the cell pellets collectedfor RNA.

At week 7 post treatment, the remaining tumor samples were collected forflow cytometry with CD44, CD24 and Hoechst staining. Remaining samplewas used for RNA extraction.

Single Cell Suspension and Flow Cytometry Staining

1. Tumors were collected in 5 mL of DMEM supplemented with 2.5% FCS in15 mL tubes.

2. Tumors were then weighed individually to determine the amount ofcollagenase to add. NOTE: use 1 mg of collagenase/1 g of tumor,concentration of collagenase=100 mg/mL in DMEM.

3. Tumors were chopped up finely using surgical blades in a petri dishand then transferred back into 15 mL tube with appropriate amount ofmedia e.g. if tumor was 1 g, then the media is topped up to 10 mL and100 μL of collagenase stock is added.

4. Samples were then incubated at 37° C. for 1 hour with shaking/tippingof tubes every 5 min.

5. After 1 hour, samples were then spun down at 500×g for 5 min. at roomtemperature.

6. Cells were then resuspended in 10 mL of 2.5% FCS+DMEM and filteredusing a 0.2 □m filter into a 50 mL tube.

7. Cells were then counted using trypan blue staining.

8. A total number of 2×10⁵ cells were stained for CD44-APC, CD24-PE andHoechst.

Staining was done with 100 mL of CD44-APC (1:50), CD24 (1:50) andHoechst (1:1000), in the dark in 1.5 ml Eppendorf tubes with rotationfor 35 min. at 4° C.

Cells were then washed with 1 mL of FACS buffer (1% FCS+PBS) andresuspended in 100 μL into small FACS tubes for acquisition.

Flow cytometry was done using LSR II at JCSMR, ANU.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

What is claimed is:
 1. A method for inhibiting proliferation, survivalor viability of a cancer stem cell (CSC) in a human subject, the methodcomprising contacting the CSC with a proliferation-, survival- orviability-inhibiting amount of a lysine specific demethylase-1 (LSD1)inhibitor, wherein the CSC has been determined to be CD44^(high),CD24^(low), and to express Sox2 mRNA or SOX2 polypeptide at a level thatis less than ⅕ of the level of Sox2 mRNA or SOX2 polypeptide of apluripotent stem cell.
 2. A method according to claim 1, wherein the CSChas been determined to express the pluripotent stem cell marker Oct4 ata level that is less than ⅕ of the level of Oct4 on a pluripotent stemcell.
 3. A method according to claim 1, wherein the CSC has beendetermined to express one or more CSC markers selected from ABCB5,ALDH1, ABCG2, α6 integrin, α2 β1 integrin, β-catenin activity, CD15,CD13, CD20, CD24, CD26, CD29, CD44, CD90, CD133, CD166, CD271, c-Met,Hedgehog, Gli, Nestin, CXCR4, LGR5, Trop2, Nodal and Activin.
 4. Amethod according to claim 1, wherein the CSC is a breast CSC.
 5. Amethod according to claim 1, wherein the CSC is a hormone receptornegative CSC.
 6. A method according to claim 1, wherein the LSD1inhibitor is a selective LSD1 inhibitor.
 7. A method according to claim1, wherein the LSD1 inhibitor is a Pan-LSD inhibitor.
 8. A methodaccording to claim 1, further comprising detecting overexpression of aLSD1 gene in the CSC prior to contacting the CSC with the LSD1inhibitor, wherein the overexpression is relative to the expression ofthe LSD gene in a non-CSC tumor cell.
 9. A method for treating a cancerin a human subject, wherein the cancer comprises cancer stem cells(CSCs) that have been determined to be CD44^(high), CD24^(low) and toexpress Sox2 mRNA or Sox2 polypeptide at a level that is less than ⅕ ofthe level of Sox2 mRNA or Sox2 polypeptide of a pluripotent stem celland non-CSC tumor cells, the method comprising administering to thesubject an LSD1 inhibitor in an effective amount to inhibit theproliferation, survival or viability of the CSCs, thereby treating thecancer in the subject.
 10. A method according to claim 9, furthercomprising identifying that the subject has or is at risk of developinga cancer comprising CSCs and non-CSC tumor cells prior to theadministration of the LSD1 inhibitor.
 11. A method according to claim 9,wherein the cancer is breast cancer.
 12. A method according to claim 9,wherein the CSCs are capable of giving rise to non-CSC tumor cells thatare hormone-resistant.
 13. A method according to claim 12, wherein thenon-CSC tumor cells are hormone receptor negative for at least onehormone receptor selected from an estrogen receptor (ER) and aprogesterone receptor (PR).
 14. A method according to claim 9, furthercomprising detecting overexpression of an LSD1 gene in a tumor sampleobtained from the subject, wherein the overexpression is relative to theexpression of the LSD gene in a non-CSC tumor cell, and wherein thetumor sample comprises CSCs that have been determined to be CD44^(high)CD24^(low) and to express Sox2 mRNA or Sox2 polypeptide at a level thatis less than ⅕ of the level of Sox2 mRNA or Sox2 polypeptide of apluripotent stem cell prior to administering the LSD inhibitor to thesubject.
 15. A method for treating or preventing a cancer in a humansubject, wherein the cancer comprises cancer stem cells (CSCs) that havebeen determined to be CD44^(high), CD24^(low) and to express Sox2 mRNAor Sox2 polypeptide at a level that is less than ⅕ of the level of Sox2mRNA or Sox2 polypeptide of a pluripotent stem cell and non-CSC tumorcells, the method comprising concurrently administering to the subject aLSD inhibitor in an effective amount to inhibit the proliferation,survival or viability of the CSCs of the cancer and a cancer therapy oragent that inhibits the proliferation, survival or viability of thenon-CSC tumor cells, to thereby treat or prevent the cancer.
 16. Amethod according to claim 15, wherein the cancer therapy or agent isselected from radiotherapy, surgery, chemotherapy, hormone ablationtherapy, pro-apoptosis therapy and immunotherapy.
 17. A method accordingto claim 15, wherein the cancer therapy or agent targets rapidlydividing cells or disrupts the cell cycle or cell division.